Download Incidence, Risk Factors and Pathogenicity of Bacteria Causing

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. Blepharoconjunctivitis. Int Ophthalmol Clin 1984; 24: 65-77.
2. McCulley JP, Shine WE. Eyelid disorders: the meibomian gland, blepharitis, and contact
lenses. Eye Contact Lens 2003; 29: S93-S95.
3. Liao HR, Lee HW, Leu HS, Lin BJ, Juang CJ. Endogenous Klebsiella pneumoniae
endophthalmitis in diabetic patients. Can J Ophthalmol 1992; 27:143-147.
4. Aristoteli LP, Bojarski B, Willcox MD. Isolation of conjunctival mucin and differential
interaction with Pseudomonas aeruginosa strains of varied pathogenic potential. Exp Eye
Res 2003; 77: 699-710.
5. Kaye S, Tuft S, Neal T, Tole D, Leeming J, Figueiredo F, et al. Bacterial susceptibility to
topical antimicrobials and clinical outcome in bacterial keratitis. Invest Ophthalmol Vis
Sci 2010; 51: 362-368.
6. Bialasiewicz A, Shenoy R, Thakral A, Al-Muniri A, Shenoy U, Al-Mughairi Z. Microbial
keratitis: a 4 year study of risk factors and traditional/complementary medicine in Oman.
Ophthalmologe 2006; 103: 682-687.
7. Lam DS, Houang E, Fan DS, Lyon D, Seal D, Wong E. Incidence and risk factors for
microbial keratitis in Hong Kong: comparison with Europe and North America. Eye
(Lond) 2002;16: 608-618.
8. Bourcier T, Thomas F, Borderie V, Chaumeil C, Laroche L. Bacterial keratitis:
predisposing factors, clinical and microbiological review of 300 cases. Br J Ophthalmol
2003; 87: 834-838.
9. Green M, Appel A, Stapleton F. Risk factors and causative organisms in microbial
keratitis. Cornea 2008; 27: 22-27.
20
10. Furlanetto RL, Andreo EG, Finotti IG, Arcieri ES, Ferreira MA, Rocha FJ. Epidemiology
and etiologic diagnosis of infectious keratitis in Uberlandia, Brazil Eur J Ophthalmol
2010; 20: 498-503.
11. Keay L, Edwards K, Naduvilath T,
Taylor HR,
Grant R, Forde SK, Stapleton F.
Microbial keratitis predisposing factors and morbidity. Ophthalmol 2006; 113: 109-116.
12. Shah VM, Tandon R, Satpathy G, Nayak N, Chawla B, Agarwal T, et al. Randomized
clinical study for comparative evaluation of fourth-generation fluoroquinolones with the
combination of fortified antibiotics in the treatment of bacterial corneal ulcers. Cornea
2010; 51: 362-368.
13. Mah-Sadorra JH, Yavuz SG, Najjar DM, Laibson PR, Rapuano CJ, Cohen EJ. Trends in
contact lens-related corneal ulcers. Cornea 2005; 24: 51-58.
14. Sueke H, Kaye S, Neal T, Murphy C, Hall A, Whittaker D, et al. Minimum inhibitory
concentrations of standard and novel antimicrobials for isolates from bacterial keratitis.
Invest Ophthalmol Vis Sci 2010; 51: 2519-2524.
15. Fleiszig SM. The Glenn A. Fry award lecture 2005. The pathogenesis of contact lensrelated keratitis. Optom Vis Sci 2006; 83: 866-873.
16. Pachigolla G, Blomquist P, Cavanagh HD. Microbial keratitis pathogens and antibiotic
susceptibilities: a 5-year review of cases at an urban county hospital in north Texas. Eye
Contact Lens 2007; 33: 45-49.
17. Passos RM, Cariello AJ, Yu MC, Höfling-Lima AL. Microbial keratitis in the elderly: a
32-year review. Arq Bras Oftalmol 2010; 73: 315-319.
21
18. Synder RW, Hyndiuk RA. Mechanisms of bacterial invasion of the cornea. In: Tasman W,
Jaeger EA, editors. Duane‘s Foundations of Clinical Ophthalmology. Philadelphia: J B
Lippincott & Co.; 1990. pp. 11-44.
19. Reichart R, Stern GA. Qualitative adherence of bacteria to human corneal epithelial cells.
Arch Ophthalmol 1984; 102: 1394-1395.
20. Panjwani N, Clerk B, Cohen M, Barza M, Baum J. Differential binding of Ps. aeruginosa
and Staph. aureus to corneal epithelium in culture. Invest Ophthalmol Vis Sci 1990; 31:
696.
21. Bancroft, J.D., A. Stevens and D.R. Turner, 1996. Theory and Practice of Histological
Techniques. 4th Edn, Churchill, Livingston, New York, London.
22. Houang E, Lam D, Fan D, Seal D. Microbial keratitis in Hong Kong:relationship to
climate, environment and contact lens disinfection. Trans R Soc Trop Med Hyg 2001; 95:
361-367.
23. Hooi SH, Hooi ST. 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