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Incidence, Risk Factors and Pathogenicity of Bacteria Causing Infectious Keratitis in Qassim Province معدل األصابة و عوامل الخطر و القدرة األمراضية للبكتيريا المسببة اللتهاب القرنية المعدى فى القصيم Abstract: Aim: This work was aimed to study the incidence and risk factors of the bacteria causing infectious keratitis among patients in Qassim province of Saudi Arabia. Furthermore, isolation of bacteria and studying its pathogenicity through experimental infection was also performed. Materials and methods: One hundred patients suffering from keratitis were subjected to clinical examinations. A total of 115 corneal swabs from these cases were collected under aseptic conditions for bacteriological examinations. The isolated bacteria were tested for pathogenicity by intraperitoneal inoculation in different groups of rabbits through the evaluation of serum biochemistry and histopathological examinations. Results: Culture of the corneal swabs revealed Pseudomonas aeruginosa, Staphylococcus aureus and unclassified bacteria as 25.21 %, 15.65 % and 13.91 % respectively. On the other hand, 52 swabs of infectious keratitis cases (45.22 %) were negative to bacteria. In the experimental study, the rabbits injected with pseudomonas became very sick within 3-4 days. The infected rabbits showed alterations in both liver and kidney functions that varied with the type of bacteria injected. Histopathologically, the internal organs of the experimented rabbits showed inflammatory reactions with degenerative changes and/or necrosis while the cornea 1 revealed edema and leukocytic infiltration. The microscopic findings were varied in severity according to the type of the bacteria. Conclusion: It could be concluded that, infectious keratitis was mostly due to Pseudomonas aeruginosa and Staphylococcus aureus. These bacteria revealed pathologic lesions and disturbances in the functions of liver and kidneys of experimented rabbits. Therefore, strict measures are recommended to control and treat infectious keratitis to avoid visual complications and systemic disturbances among infected patients. Key Words: Infectious keratitis, Pseudomonas aeruginosa, Staphylococcus aureus معدل األصابة و عوامل الخطر و القدرة األمراضية للبكتيريا المسببة اللتهاب القرنية المعدى فى القصيم 2 يوسف حمود الدباسى ، 1صالح الدين مصيلحى على 4 محمد اعجاز أحمد ، 3أمجد على خان 1-3قسم البصريات – كلية العلوم الطبية التطبيقية – جامعة القصيم 2-4 قسم المختبرات الطبية -كلية العلوم الطبية التطبيقية – جامعة القصيم الملخص العربى الغرض :كان الغرض من هذا العمل هو دراسة معدل االصابة وعوامل الخطر للبكتيريا المسببة اللتهاب القرنية المعدى فى مرضى منطقة القصيم بالمملكه العربية السعودية .هذا باألضافة الى عزل تلك البكتيريا ودراسة القدرة االمراضية لها من خالل العدوى التجريبية. المواد وخطة العمل :تم فحص اكلينيكى لعدد 011مريض يعانون من التهاب القرنية .وقد جمعت 001مسحة من القرنية من هذه الحاالت تحت ظروف معقمة للفحص البكتيرولوجى .وقد اختبرت البكتيريا المعزولة لقدرتها االمراضية من خالل الحقن البريتونى لمجموعات متعددة من االرانب وذلك باالستعانة بفحص المصل من الناحية الكيميائية الحيوية وكذلك بالفحص النسيجى المرضى. 2 النتائج :اسفر فحص المسحات القرنية عن عزل ميكروب السدومونس ايروجينوزا والمكورات العنقودية وبعض البكتيريا الغير مصنفة نسبة % 01.51 ، % 51.50و % 09.30على التوالى .وعلى النحو األخر فان % 21.55من مسحات القرنية اسفرت عن نتائج سلبية بالنسبة للعزل البكتيرى. وقد اسفرت الدراسة التجريبية ان االرانب المحقونة بالسيدومونس اصبحت مريضة خالل 2-9ايام من الحقن .كما اظهرت االرانب المصا بة تجريبيا عن تغيرات فى وظائف الكبد والكلى والتى تباينت مع نوع البكتيريا المستخدم فى الحقن اما الفحص النسيجى المرضى فقد تبين تفاعالت التهابية وتغيرات انهدامية ونخر باالعضاء الداخلية لالرانب المجربة اما القرنية فقد ظهر بها وذمة وارتشاحات خلوية .وقد تباينت تلك التغيرات الميكروسكوبية حسب نوع البكتيرايا المستخدم فى العدوة التجريبية. الخالصة :خلصت الدراسة الى ان التهاب القرنية المعدى تسبب غالبا بميكروب السدومونس ايروجينوزا والمكورات العنقودية .وقد احدثت هذه البكتيرا افات مرضية وخلل فى وظائف الكبد والكلى لالرانب المجربة .ولذلك يوصى بأتخاذ تدابير صارمة لليسطرة وعالج التهاب القرنية المعدى وذلك لتجنب المضاعفات واالختالل العضوى بين المرضى المصابين. Introduction: Ocular surface of healthy individuals inherently supports a small population of naturally inhabitant bacteria as coagulase negative staphylococci (CNS) which have been found to exist as Several ocular disorders are associated with 2 commensals on the mucosa and lid margins.1, different Gram positive and Gram negative bacteria, including Staphylococcus aureus, Streptococcus sp., Bacillus subtilis, Rhodococcus sp., Pseudomonas aeruginosa, Haemophilus influenzae, Haemophilus aegyptius, and Klebsiella species.3, 4 Microbial keratitis is usually used to describe various types of corneal infections but in most cases, it is used to describe bacterial keratitis because the other types of keratitis are named by the specific causative agent as acanthamoeba, fungi, or viruses. Severe keratitis can potentially lead to blindness and in many cases surgical intervention are needed.5 Microbial keratitis spreads 3 commonly, in spite of many advances in diagnosis, management and availability of potent antibiotics. Common use of contact lenses, ocular surface diseases, corneal trauma, use of immunosuppressive medications and ocular surgery like corneal graft are different types of factors which cause bacterial keratitis.6 The contact lens wearing is the leading cause of keratitis in some developed countries while trauma and ocular surface disease are the leading causes in other countries. Contact lens wearing is one of the greatest risk factors for infective keratitis and may account for 20 percent to 50 percent of all cases.5, 7-12 Contact lens related bacterial keratitis are usually caused by Pseudomonas species.5, 8,11,13,14 These bacteria are very virulent and can be visually devastating because of their ability to alter genes which are related to virulence, survival, and adaptation.15 Staphylococcus aureus, Streptococcus pneumoniae and Pseudomonas species are the common types of bacteria causing corneal ulcers worldwide. 9, 16, 17 Pseudomonas aeruginosa is the most common and virulent ocular pathogen and has the ability to perforate the cornea in just 72 hours. Staphylococcus aureus, Streptococcus pneumonia and Pseudomonas aeruginosa have the ability to penetrate the stroma even with adequate host-defenses.18-20 The present study aimed to investigate the incidence and risk factors of infectious keratitis together with isolation and identification of the causative bacteria. Furthermore, to investigate the pathogenesis of the isolated and identified bacteria on different groups of rabbits through experimental infection by studying liver and kidney function parameters and histopathological examinations. 4 Materials and methods: This was a cross sectional study conducted during the period of December 2010 to May 2011 in collaboration with King Fahd Specialist Hospital Buraidah, which is a tertiary care hospital in Qassim province. Patients coming directly to emergency department or referred from peripheral basic health units or ophthalmologists were included in this research project. Patients suffering from keratitis underwent ophthalmic examination and complete interview was done about the cause of keratitis. Hundred Patients were received with painful red eyes and subjected to detailed clinical examination including visual acuity, corneal epithelial defects, number and position of corneal infiltrates and anterior chamber reaction. Sampling: Under aseptic conditions, 115 corneal swabs/scrapings were obtained form 100 patient’s suspected of bacterial keratitis for bacteriological examinations. The samples were transferred immediately to the laboratory for processing. Bacterial isolation and identification: This technique was done routinely in all collected samples including culturing, sub-culturing and purification, isolation and identification. The collected swabs were inoculated in tryptic soya broth overnight at 37˚ C. Consequently, the broth was inoculated onto blood agar, MacConkey’s agar, mannitol salt agar, and chocolate agar media, and then incubated aerobically at 37˚C for maximum up to 48 hours. Inoculated chocolate agar plates were left in anaerobic incubator at 5% CO2. All the bacterial isolates were identified by their colony morphology, Gram staining, 5 pigment production, relevant biochemical tests and API strips according to the manufacturer. The minimum criteria for the positive culture were considered upon the growth of three colonies on one solid medium. Experimental study. Animals. This experimental study was conducted on 27 male albino rabbits weighing between 2.0 - 2.5kg, obtained from farm of Qassim University. The rabbits were properly examined and proved free from any disease. The rabbits were acclimatized for 7 days in the animal house conditions before starting the experiment. The experimental animals were housed in air conditioned rooms at 2123oC and 60-65% relative humidity and kept on 12 h light/dark cycle. They were housed in standard aluminum cages and fed with standard rabbit diet and normal tap water. They were handled as per the international rules implemented in the experimental laboratory animals, College of Applied Medical Sciences, Qassim University. Experimental Groups and Protocol. The Rabbits were divided randomly into three equal groups, each containing 9 rabbits. Group 1 : Rabbits as negative control group were inoculated intraperitoneally (IP) with sterile buffer saline. 6 Group II : Rabbits were IP inoculated with isolated Pseudomonas aeruginosa. Group III : Rabbits were IP inoculated with isolated Staphylococcus aureus. Bacterial inoculums were prepared by cultivating each bacterial species onto nutrient agar for 24 h at 37 oC, and then 5-7 colonies were transferred to a tube containing 5 ml sterile normal saline solution. The tubes were vortexed to make a bacterial suspension with turbidity equal to 0.5 McFarlands standard solution (equivalent to 5 - 108 colony-forming units [CFU]/mL). The suspension was then adjusted to a final concentration of 105 CFU/mL, as verified by a quantitative bacterial count on Mueller-Hinton agar plates. A bacterial suspension of 2 ml from each sample was carefully injected intraperitoneally to each experimental animal using sterilized syringes. Blood and tissue collection: Rabbits were observed two weeks for any clinical signs of illness. Blood samples were collected by a syringe from the lower border of the pinna using xylene as a vasodilator. Blood sample were taken for biochemical analysis after 3, 7 and 14 days post-infection. Besides this different tissues were taken for histopathological examination at 7 and 14 days post-infection. Biochemical analysis: Blood samples (serum) were monitored for liver and kidney function analysis. Diagnostic kits for serum ALT, AST, urea, uric acid and creatinine were purchased from Human Diagnostic Kits (Human GmbH, Wiesbaden Germany) and were used as per manufacturer’s instructions. Histopathological techniques: 7 Tissue sections from the internal organs (eyes, liver and kidneys) of experimental rabbits were taken and immediately fixed in 10% neutral buffered formalin, then dehydrated in increasing concentrations of ethyl alcohol, cleared in xylene, blocked in paraffin and sectioned as 5 µm using rotary microtome. The obtained tissue slides were stained with hematoxylin and eosin21 Statistical analyses: One-way ANOVA was used to evaluate the significant difference of the different treatments and duration. A probability at level of 0.05 or less was considered significant. Means and standard errors were also estimated. All statistical analyses were run on the computer, using the SAS program (SAS, 2003). Results: Clinical characteristics in human subjects A total of 100 patients (115 eyes) with a diagnosis of bacterial keratitis were examined during the study period of 6 months. Out of 100 subjects, 85 cases were examined for the first time in the emergency department of our hospital whereas 15 cases were referred by general practitioners or ophthalmologists. The age of the patients ranged from 12 years to 55 years (mean age 21 years). Sex distribution was 23 men and 77 women. Most of patients were living in urban areas. 8 Frequency of predisposing ocular conditions. The examined patients revealed that, contact lens (CL) wearing was the most common risk factor and was found in 51 eyes (44%) and no risk factors were identified in 16% of cases. All the predisposing factors are summarized in Table 1. Diabetes mellitus was a risk factor in 20 cases but there was no significant correlation between systemic risk factors and severity of clinical presentation. The clinical course of these patients was acute with lid and conjunctival edema, reduced vision, pain, redness, severe photophobia and discharge. Table 1: The recorded predisposing factors among patients suffering from infectious keratitis. Incidence (%) Number of swabs (n=115) Contact lens wear 44 51 Ocular surface disease 11 13 Corneal trauma 22 25 Corneal surgery 07 08 None 16 18 Factor Table-1 describes that the contact lens wearing was the most common risk factor among patients of infectious keratitis followed by corneal trauma. Right eye was observed to be more involved by keratitis (57 %) in 66 cases as compared to left eye in (43 %) 49 cases. Infection was bilateral in fifteen patients (13%). Visual acuity at time of examination ranged from 20/20 to 20/200. Nasal infiltrates were most common (41%). Corneal infiltrates were single in 84 eyes (73%) and multiple in 31 (27%). In addition to this, anterior chamber inflammation was absent in 41.3% (47) of cases. A 1+ to 2+ Tyndall effect was present 9 in 57% (66) of cases, whereas severe anterior chamber inflammation (3+ to 4+) and hypopyon were present in 2 and 1.7% of cases, respectively. Furthermore, the eyes showed severe anterior chamber reaction and no of corneal infiltrates from which Gram negative bacteria were isolated. The location of the infiltrates was distributed as shown in Table 2. Table 2: The location of the corneal infiltrate among patients suffering infectious keratitis Position of corneal infiltrates Incidence (%) Temporal 36 Nasal 41 Central 15 Diffuse 8 Microbiological characteristics Culture results of the collected corneal scraping swabs revealed the isolation of bacteria from 63 swabs (54.78%), where Pseudomonas aeruginosa, Staphylococcus aureus and unclassified bacteria were isolated with a percentage of 25.21, 15.65 and 13.91 %; respectively. On the other hand, 52 swabs of infectious keratitis cases (45.22%) were negative to bacteria. Also multiple 10 organisms were found in 8 (14%) swabs from corneal scraping and the Gram negative were the predominant type of bacteria. In contact lens wearing group, 26 (59.09%) of the corneal scrapings were positive and the cultured bacteria were Pseudomonas aeruginosa in 21(47.72%) of cases. The percentage of Gram positive bacteria was 5 (45.45%) in the ocular surface disease group and 8 (36.36%) in the corneal trauma group. Mixed bacteria not fitted in specific group were also cultured in 16 (13.91%).The bacterial spectrum is shown in Table 3. Table 3: Frequency distribution of bacteria isolated from corneal swabs of patients suffering from infectious keratitis. Type of microorganisms Number (Percentage) Gram positive cocci (Staphylococcus aureus) 18 (15.65%) Gram negative bacilli (Pseudomonas aeruginosa) 29 (25.21%) Unclassified bacteria 16 (13.91%) Total bacterial isolates 63 (54.78%) Clinical findings in experimental models: The intraperitoneal injection of rabbits with either Psudomonas aeruginosa or Staphylococcus aureus caused lethargy, pain and diarrhea. Clinically, the rabbits injected intraperitoneally with Pseudomonas aeruginosa became very sick within 3-4 days while those infected with Staphylococcus aureus, were apparently normal. 11 Biochemical profile: As shown in Table 4, the level of urea increased to a higher extent in rabbits within 3 days of the intraperitoneal injection of Staphylococcus aureus and Pseudomonas aeruginosa. The level of urea was increased to a higher extent after two weeks of intraperitoneal injection with Staphylococcus as compared to Pseudomonas injection. There was no marked change in the level of uric acid in all groups that intraperitoneally injected with either S. aureus or P. aeruginosa. There was also no much variation in the level of creatinine in all the groups of rabbits. ALT and AST values were also within limits of reference in all the groups of rabbits injected with either S. aureus or P. aeruginosa. Table 4: Different biochemical parameters in Rabbits intraperitoneally infected with bacteria for the evaluation of liver and kidney function parameters. Time / day 3 Intraperitoneal injection Urea (mg/dL) Uric acid (mg/dL) Creatinine (mg/dL) ALT (U/L) AST (U/L) Control 29.53±3.54 0.64±0.08 0.87±0.12 27.92±1.45 14.59±1.21 43.81±3.23* 0.24±0.01 0.578±0.11 26.33±1.26 16.34±1.87 42.35±2.47* 1.03±0.06 0.84±0.20 32.05±2.21 13.32±1.22 15.09±1.98 0.53±0.09 1.08±0.02 24.75±3.10 9.20±1.98 7.46±1.68 1.84±0.04 1.92±0.07 27.87±2.14 12.22±1.20 18.33±2.13 0.76±o.08 1.01±0.08 27.92±1.18 14.87±2.13 Control 31.96±3.45 0.42±0.07 0.77±0.04 34.87±1.76 15.87±2.76 Pseudomonas aeruginosa 26.28±2.87 0.24±0.03 0.94±0.12 29.19±2.25 13.73±1.54 Pseudomonas aeruginosa Staphylococcu s aureus Control 7 14 Pseudomonas aeruginosa Staphylococcu s aureus 12 Staphylococcu s aureus 50.46±.86* 0.24±0.08 0.67±0.015 23.80±2.12 8.88±0.34 Values are given as ±SD for groups of 9 rabbits. Values are statistically significant *p<0.05 compared to control, ALT (alanine aminotransferase), AST (aspartate aminotransferase) Histopathology: Rabbits IP inoculated with Pseudomonas aeruginosa: At7th day of IP injection, oedema in the corneal subepithelial tissue and congestion, vacuolar degeneration as well as pleomorph-nuclear leukocytes in the liver and kidneys were evident (Fig. 1). At 14th day of IP injection, the corneal stroma was oedematous and infiltrated with pleomorph-nuclear leukocytes (Fig. 2). The liver showed cloudy swelling in the hepatocytes while the kidneys exhibited tubular nephrosis mainly vacuolar degeneration in the renal tubules. Rabbits IP inoculated with Staphylococcus aureus: Fig. 1: Rabbit kidney showing congestion, vacuolar degeneration and pleomorphnuclear leukocytes inoculated with Pseudomona aeruginosa. 13 Fig. 1: Rabbit’s kidney IP inoculated with Pseudomonas aeruginosa at7th day of injection, showing congestion, vacuolar degeneration as well as pleomorph-nuclear leukocytes. H & E stain, X 400. Fig. 2: Rabbit eye showing corneal stromal edema and pleomorphnuclear leukocytic infiltration inoculated with Pseudomonas aeruginosa. Fig. 2: Rabbit’s eye IP inoculated with Pseudomonas aeruginosa at14th day of injection, showing corneal stromal edema and pleomorph-nuclear leukocytic infiltration. H & E stain, X 250. 14 At7th day of IP injection, focal desquamation in the corneal epithelium was evident with pleomorph-nuclear leukocytes in the stroma, this in addition to congestion, vacuolar degeneration, coagulative necrosis and pleomorph-nuclear leukocytes in the liver and kidneys (Fig. 3). At 14th day of IP injection, proliferation of corneal epithelium and pleomorph-nuclear leukocytes infiltration in the corneal stroma was observed (Fig. 4). Vacuolar degeneration and focal necrosis were seen in the hepatocytes while the kidney revealed cloudy swelling in the renal epithelium. Fig. 3: Rabbit liver showing congestion, vacuolar degeneration, coagulative necrosis and pleomorphnuclear leukocytes inoculated with Staphylococcus aureus. Fig. 3: Rabbit’s liver IP inoculated with Staphylococcus aureus at7th day of injection, showing congestion, vacuolar degeneration, coagulative necrosis and leukocytic infiltration. H & E stain, X 400. 15 inoculated with Staphylococcus aureus. Fig. 4: Rabbit’s eye IP inoculated with Staphylococcus aureus at14th day of injection, showing proliferation of corneal epithelium and leukocytic infiltration in the corneal stroma. H & E stain, X 250. Discussion: In the present study, the incidence of culture positive microbial keratitis was 54.78% among the population of Qassim province. Among the isolated bacteria, Pseudomonas aeruginosa was the most common cultured bacteria followed by staphylococcus aureus. Parallel studies in other regions of the world, conducted on large scales have shown varying degrees of culture positive bacteria. Pachigolla et al 2007 showed that, eyes of 131 patients underwent 139 corneal scrapings presumed microbial keratitis. They also observed that, Pseudomonas aeruginosa isolated from 73 cases (52.5%). 16 In a study conducted by Passos et al. showed culture positivity of 53.5% organisms (bacteria 47.0 %, fungi 6.1 %, and acanthamoeba 0.4 %). The most frequent bacteria were the Gram-positive cocci (mostly coagulase-negative, Staphylococci) and Gram-negative bacilli (mostly the genera Pseudomonas, Moraxella and Proteus). Green et al. showed that, cultures of corneal scrapings were positive in 65% of cases, where Pseudomonas aeruginosa (44; 17%), coagulase-negative staphylococci (22; 9%), Staphylococcus aureus (19; 8%), and fungi (7; 3%) were commonly recovered. In our study, Pseudomonas aeruginosa was the most common bacteria cultured (25.21%) and similarly the above mentioned studies also show that this bacteria is the most common cultured followed by Staphylococci although the culture positive rate for pseudomonas is higher as compared to our study. This variation may be due to different risk factors in these studies.9, 13, 14 A high rate of Pseudomonas aeruginosa isolation from contact lens related bacterial keratitis was also found in some other countries like Southeast Asia 52 % in Hong Kong, 64% in Malaysia and 79% in Singapore. 22-24 Tremendous use of contact lens during the last decade increased the risk factor of microbial keratitis dramatically. This was also observed in our study (44%), it contributed to almost 50% cases of culture positive microbial keratitis for Pseudomonas aeruginosa. Corneal surface diseases and corneal trauma have been found to be second most common cause of bacterial keratitis, accounting for 33% of cases. Willcox, 2007 also observed that, Pseudomonas aeruginosa is usually the most common bacterial pathogen isolated from cases of keratitis. This infection poses a serious threat to normal vision and is associated with extended wear of contact lenses and eye trauma.25 Tissue damage during bacterial keratitis results from the action of 17 bacterial products on ocular tissues and from the host inflammatory response to the infection. Staphylococcus keratitis can result in irreversible corneal scarring, resulting in a loss of visual acuity.26 In the present study, intraperitoneal inoculation of rabbits with Pseudomonas aeruginosa showed degeneration of corneal epithelia with oedema and congestion in the subepithelial tissue at 7th day of inoculation while at 14th day, the stroma infiltrated with pleomorph-nuclear leukocytes. Gray and Kreger have also shown that Pseudomonas aeruginosa infections cause acute inflammation and liquefaction necrosis of the cornea.27 Different types of bacterial and other microbial infections in humans alter the homeostasis by producing different types of toxins. The primary function of the liver and kidneys is to expel these different types of toxins that may also be produced as a result from the body's metabolism of food and other types of infections. So, malfunction of the liver and kidney can translate to serious conditions that may be life threatening. Kidneys act as a filtering system by getting rid of different waste products. They balance the body's fluid content by reabsorbing immense amounts of water into the blood, produce hormone that helps to make red blood cells and help to control blood pressure. As shown in Table 4, the level of urea increased to a higher extent within 3 days of the intraperitoneal injection of Staphylococcus aureus and Pseudomonas aeruginosa. The level of urea was increased to a higher extent after two weeks of intraperitoneal injection with Staphylococcus as compared to Pseudomonas injection. The increase in urea level could be due to the recorded histopathological lesions in both kidneys and liver. 18 In the current study, IP inoculation of rabbits with Pseudomonas aeruginosa or Staphylococcus aureus revealed edema or desquamation of corneal epithelium with pleomorphnuclear leukocytes in the corneal stroma at 7th day PI while at 14th day PI, infiltration of pleomorph-nuclear leukocytes in the corneal stroma and proliferation of corneal epithelium were evident. O’Callaghan et al., has also mentioned that, Staphylococcus aureus corneal infection results in extensive inflammation and tissue damage.28 On the other hand, the liver and kidneys of rabbits intraperitoneally inoculated with Pseudomonas aeruginosa or Staphylococcus aureus showed inflammatory reactions and degenerative as well as necrotic changes at 7 days while at 14 days of experiment, degenerative changes and focal necrosis were evident. Tzanakakis et al., has also reported that, P. aeruginosa infection causing necrosis of hepatic parenchyma.29 Another studies showed that, Pseudomonas aeruginosa infection, in acute cases, cause a systemic infection due to Gram-negative bacterial septicemia While, in the subacute to chronic stages of the disease, multifocal necrosis with abscessation may be present in kidneys. 30 Conclusion: As infectious keratitis represents a potentially significant threatening ocular disease, initial diagnosis facilitates its resolution in hopes of preventing future visual loss. Our study concluded that, 54.78% of microbial keratitis among patients in Qassim province was caused by bacteria mainly Pseudomonas aeruginosa and Staphylococcus aureus. These infections could induce pathologic lesions in the liver and kidneys and consequently alterations in some biochemical parameters. Therefore, strict measures are recommended to control and treat infectious keratitis. 19 References: 1. McCulley JP. 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Culture proven bacterial keratitis in a Malaysian general hospital. Med J Malaysia 2005; 60: 614-623. 24. Tan DT, Lee CP, Lim AS. Corneal ulcers in two institutions in Singapore: analysis of causative factors, organisms and antibiotic resistance. Ann Acad Med Singapore 1995; 24: 823-829 25. Willcox MD. Pseudomonas aeruginosa infection and inflammation during contact lens wear: a review. Optom Vis Sci 2007; 84: 273-278. 22 26. Hazlett LD, Zucker M, Berk RS. Distribution and kinetics of the inflammatory cell response to ocular challenge with Pseudomonas aeruginosa in susceptible versus resistant mice. Ophthalmic Res 1992; 24: 32-39. 27. Gray LD, Kreger AS. Rabbit corneal damage produced by Pseudomonas aeruginosa infection. Infect Immun 1975; 12: 419-432. 28. O’callaghan RJ, Callegan MC, Moreau JM, Green LC, Foster TJ, Hartford OM, et al. Specific Roles of Alpha-Toxin and Beta-Toxin during Staphylococcus aureus Corneal Infection Infec Immun 1997; 5: 1571-1578. 29. Tzanakakis GN, Veronikis DK, Anastasiou ED, McCully KS, Dimitracopoulos G. Histopathological lesions produced by P. aeruginosa lipopolysaccharide in rats. J Exp Pathol 1989; 4:199-211. 30. Percy DH, Barthold SW. Pathology of Laboratory Rodents and Rabbits. 1993; 1: 37-38. 23 Figure legends: Fig. 1: Kidney, of rabbits IP inoculated with Pseudomonas aeruginosa at7th day of IP injection, showing congestion, vacuolar degeneration as well as pleomorph-nuclear leukocytes. H & E stain, X 400. Fig. 2: Eye, of rabbits IP inoculated with Pseudomonas aeruginosa at14th day of IP injection, showing corneal stromal edema and pleomorph-nuclear leukocytic infiltration. H & E stain, X 250. Fig. 3: Liver, of rabbits IP inoculated with Staphylococcus aureus at7th day of IP injection, showing congestion, vacuolar degeneration, coagulative necrosis and pleomorph-nuclear leukocytes. H & E stain, X 400. Fig. 4: Eye, of rabbits IP inoculated with Staphylococcus aureus at14th day of IP injection, showing proliferation of corneal epithelium and pleomorph-nuclear leukocytes infiltration in the corneal stroma. H & E stain, X 250. 24