Isolation and identification of aerobic bacteria associated with eye infection in human

89 Pages·2004·0.55 MB·English

Isolation and identification of aerobic bacteria associated
with eye infection in human

By
Salma Abdul Rahman Mohammed Masri
B.Sc. Faculty of Science
University of Khartoum

Thesis submitted for the partial fulfillment of the requirements for the
Degree of Master of Science (M.Sc) in Microbiology

Supervisor:
Dr. Awad A.Ibrahim
BV. Sc. University of Khartoum
M.Sc. University of Amsterdam
PhD. State U. of Utrecht

Faculty of Veterinary Medicine
University of Khartoum

Department of Microbiology
Faculty of Veterinary Medicine
University of Khartoum
Nov. 2003

1
Dedication

To my caring, encouraging, great parents.

To my beloved, sincere sisters and brothers.

With love

2
Acknowledgments
I do appreciate the continuous guidance, patience and careful
supervision of Dr. Awad A. Ibrahim.
I would like to express my deep gratitude to Fowzia M.Hussien
and Mona Matassim for their extreme support, continuous advice
and unlimited help in lab work arrangements.
I am also thankful to the staff of Khartoum Educational Eye
Hospital and Omdurman Educational Hospital for their great
assistance during sample collection.
I am deeply indebted to all staff of the Department of
Microbiology, Faculty of Veterenary Medicine, U.K for being
extremely efficient in bringing out this work.
Thanks also extended to the technicial staff of the media
preparation lab for the great facilities and endless help.
I am deeply appreciating the warm feelings, and unforgettable
kindness of my friends and colleagues.
Special thanks to my sister; Mai Almasri who proved to be an
editor of a remarkable patience and valuable guidance.
Finally, I am thankful to my family for the greatest gift I could
ever receive, the touch of care and sense of love.

3
Abstract
This study was concerned primarily with the isolation and
identification of aerobic bacteria associated with human ocular infection in
Khartoum State. Eye swabs were collected from 68 patients of different age
groups including infants, children and adults. The collection of specimens
was made during the period from September to November (2002) and from
January to March (2003).
Bacteria were isolated from (55.8%) of total number of the samples
collected from patients with eye infection. The isolated Gram- positive
bacteria were Staphylococci species which amounted to (60.9%) as the most
commonly cause of eye infection. Streptococci species and Corynebacterium
xerosis were also isolated. The isolated Gram-negative bacteria were
Branhamella catarahlis, Moraxella spp., Pseudomonas spp. and Klebsiella
pneumonia. Staphylococcus simulans, Staphylococcus chromogenes and
Pseudomonas fluorescens were isolated for the first time in this study from
ocular infection.
4
Antibiotic sensitivity tests were made and about (76.9%) of all
isolated bacteria were sensitive to gentamycin which was the drug of choice;
however, (79.4%) were highly resistant to ampicillin. In contrast
Streptococcus pyogenes and Streptococcus pneumoniae were found resistant
to chloramphenicol; the commonly used antibiotic for the treatment of
ocular infection.

5
ﺔﺣوﺮﻃﻷا ﺺﺨﻠﻣ
ت ﺎ(cid:1127)(cid:1127)ﺑﺎﻬﺘﻟﻻ ﺔﺒﺒ(cid:1127)(cid:1127)ﺴﻤﻟا ﺔ(cid:1127)(cid:1127)ﻴﺋاﻮﻬﻟا ﺎ(cid:1127)(cid:1127)ﻳﺮﺘﻜﺒﻟا ﻒﻴﻨ(cid:1127)(cid:1127)ﺼﺗ و لﺰ(cid:1127)(cid:1127)ﻋ ﻰ(cid:1127)(cid:1127)ﻟإ ﺔ(cid:1127)(cid:1127)ﺳارﺪﻟا ﻩﺬ(cid:1127)(cid:1127)ه ﺖﻓﺪ(cid:1127)(cid:1127)ه
ﺎ(cid:1127)ﻀﻳﺮﻡ 68 ﻦ(cid:1127)ﻡ نﻮ(cid:1127)ﻴﻌﻟا ﻦ(cid:1127)ﻡ ت ﺎﺤﺴﻡ ﺬ ﺥﺄﺑ تﺎﻨﻴﻌﻟا ﺖﻌﻤﺟ . مﻮﻃﺮﺨﻟا ﺔﻳﻻو ﻲﻓ نﻮﻴﻌﻟا
ﻦﻴﺗﺮ(cid:1127)ﺘﻓ ﻰﻠﻋ ،ﻦﻴﻐﻟﺎﺒﻟا و لﺎﻔﻃﻷاو ﻊﺽﺮﻟا ﺖﻨﻤﻀﺗ ﺔﻔﻠﺘﺨﻡ ﻪﻴﻨﺳ رﺎﻤﻋﺄﺑو نﻮﻴﻌﻟا بﺎﻬﺘﻟﺎﺑ
ﺮﺒﻤﻓﻮﻥ ﻰﻟإ ﺮﺒﻤﺘﺒﺳ ﻦﻡ ﺎﻤﻬﻟوأ ﻦﻴﺘﻔﻠﺘﺨﻡ ﻢﺗ .2003 سرﺎﻡ ﻰﻟإ ﺮﻳﺎﻨﻳ ﻦﻡ ﺎﻤﻬﻴﻥﺎﺛ و ,2002
) ﻦﻡ ﺎﻳﺮﻴﺘﻜﺒﻟا لﺰﻋ55 و ماﺮﺠﻠﻟ ﺔﻐﺒﺼﻟا ﺔﺒﺟﻮﻡ ﺎﻳﺮﻴﺘﻜﺒﻟا ﺖﻠﻤﺷ ﺚﻴﺡ ، تﺎﻨﻴﻌﻟا ﻦﻡ (%
تارﻮ(cid:1127)ﻜﻤﻟا ﻰ(cid:1127)ﻠﻋ ﺖﻠﻤ(cid:1127)ﺷ ماﺮ(cid:1127)ﺠﻠﻟ ﺔﻐﺒ(cid:1127)ﺼﻟا ﺔ(cid:1127)ﺒﺟﻮﻡ ﺎﻳﺮﻴﺘﻜﺒﻟا .ماﺮﺠﻠﻟ ﺔﻐﺒﺼﻟا ﺔﺒﻟﺎﺳ ﺎﻳﺮﻴﺘﻜﺒﻟا
نﻮ(cid:1127)ﻴﻌﻟا تﺎ(cid:1127)ﺑﺎﻬﺘﻟﻻ ﺐﺒ(cid:1127)ﺴﻡ ﺮ(cid:1127)ﺜ آأ ﺖﻠﻜ(cid:1127)ﺷ ةﺮﻴﺥ ﻷاو ،ﺔﻳدﻮﻘﻨﻌﻟا ﺎﻳﺮﻴﺘﻜﺒﻟاو تﺎﻳﺪﺗﻮﻟاو ﺔﻴﺤﺒﺴﻟا
ﺔﺒﺴﻨﺑ60.9%) ماﺮﺠﻠﻟ ﺔﻐﺒﺼﻟا ﺔﺒﻟﺎﺳ ﺎﻳﺮﻴﺘﻜﺒﻟا .ﺔﻟوﺰﻌﻤﻟا ﺎﻳﺮﻴﺘﻜﺒﻠﻟ ﻲﻠﻜﻟا عﻮﻤﺠﻤﻟا ﻦﻡ (
ﺔ(cid:1127)ﺳارﺪﻟا ﻩﺬ(cid:1127)ه ﻲ(cid:1127)ﻓ . ﻼﻴﻴ(cid:1127)ﺴﺒﻴﻠآ و سﺎﻥﻮﻡودﻮﺴﺑ ،ﻼﻠﻴﺴآارﻮﻡ ،ﺲﻴﻟارﺎﺗﺎآ ﻼﻠﻴﻡﺎﻬﻥاﺮﺑ ﺖﻠﻤﺷ
ﻲ (cid:1127)(cid:1127)هو ﻰ(cid:1127)(cid:1127)ﻟوﻷا ةﺮ(cid:1127)(cid:1127)ﻤﻠﻟ و نﻮ(cid:1127)(cid:1127)ﻴﻌﻟا تﺎ(cid:1127)(cid:1127)ﺑﺎﻬﺘﻟ ﻹ ﺔﺒﺒ(cid:1127)(cid:1127)ﺴﻤﻟا ﺎ(cid:1127)(cid:1127)ﻳﺮﻴﺘﻜﺒﻟا ﻦ(cid:1127)(cid:1127)ﻡ عاﻮ(cid:1127)(cid:1127)ﻥأ ﺔ(cid:1127)(cid:1127)ﺛﻼﺛ لﺰ(cid:1127)(cid:1127)ﻋ ﻢ(cid:1127)(cid:1127)ﺗ
سﺎﻥﻮﻡودﻮ(cid:1127)(cid:1127)(cid:1127)(cid:1127)(cid:1127)(cid:1127)(cid:1127)ﺴﺑ ،ﺲﻨﻴﺟﻮ(cid:1127)(cid:1127)(cid:1127)(cid:1127)(cid:1127)(cid:1127)(cid:1127)ﻡوﺮآ سﺎﻜآﻮآﻮﻠﻴﻓﺎﺘ(cid:1127)(cid:1127)(cid:1127)(cid:1127)(cid:1127)(cid:1127)(cid:1127)ﺳ ،ﺲﻥﻻﻮﻴﻤﻴﺘ(cid:1127)(cid:1127)(cid:1127)(cid:1127)(cid:1127)(cid:1127)(cid:1127)ﺳ سﺎﻜآﻮآﻮﻠﻴﻓﺎﺘ(cid:1127)(cid:1127)(cid:1127)(cid:1127)(cid:1127)(cid:1127)(cid:1127)ﺳ
.ﺰﺴﻨﻴﺴﻳرﻮﻠﻓ
ﺔﺒ(cid:1127)ﺴﻥ ﺖﻠﺠ(cid:1127)ﺳ ﺚ(cid:1127)ﻴﺡ ، ﺔ(cid:1127)ﻟوﺰﻌﻤﻟا ﺎ(cid:1127)ﻳﺮﻴﺘﻜﺒﻟا عاﻮ(cid:1127)ﻥ أ ﻰ(cid:1127)ﻠﻋ ﺔﻴ(cid:1127)ﺳﺎﺴﺡ تارﺎﺒﺘﺥا ﺖﻳﺮﺟأ
) ﺔﺒﺴﻨﺑ ﻦﻴﺴﻴﻡﺎﺘﻨﻴﺠﻟا رﺎﻘﻋ ﺪﺽ ﺔﻴﺳﺎﺴﺤﻟا ﻦﻡ ﺔﻴﻟﺎﻋ76.9 ﺔﻡوﺎﻘﻡ تﺮﻬﻇأ ﺔﻴﺒﻠﻏﻷاو، (%
) ﺔﺒ(cid:1127)ﺴﻨﺑ ﻦﻴﻠﻠﻴ(cid:1127)ﺴﻴﺒﻡ ﻷا رﺎﻘﻋ ﺪﺽ79.4 ﺔﻴﺤﺒ(cid:1127)ﺴﻟاو ﺔﻳﺪﻳﺪ(cid:1127)ﺼﻟا ﺔﻴﺤﺒ(cid:1127)ﺴﻟا ﺎ(cid:1127)ﻳﺮﻴﺘﻜﺒﻟا ﺎ(cid:1127)ﻤﻨﻴﺑ ، (%
بﺎ(cid:1127)ﻬﺘﻟا جﻼ(cid:1127)ﻌﻟ لﺎﻤﻌﺘ(cid:1127)ﺳ ﻻا ﻊﺋﺎﺸﻟا يﻮﻴﺤﻟا دﺎﻀﻤﻟا ؛لﻮﻜﻴﻨﻴﻔﻡارﻮﻠﻜﻠﻟ ﺔﻡوﺎﻘﻡ ﺖﻥﺎآ ﺔﻳﻮﺋﺮﻟا
.يﺮﻴﺘﻜﺒﻟا نﻮﻴﻌﻟا
6
Table of contents
Subject page

Dedication………………………………………………………………… I
Acknowledgments ………………………………………………………… II
Abstract ……………………………………………………………………. III
Arabic Abstract …………………………………………………………… V
Table of contents…………………………………………………………..… VI
List of figures……………………………………………………………...… IX
List of tables……………………………………………………………….… X
Introduction……………………………………………………………..…… 1
(Chapter one: Literature Review) ……………………………………...… 3
1.1 Basic anatomy of the Eye…………………………………...…... 3
1.2 Defense Mechanisms of the outer Eye………………………….. 4
1.3 Infection of the Eye…………………………………………….. 5
1.4 The common infectious diseases of the Eye………………….… 7
1.4.1 Conjunctiva infections (conjunctivitis)………………….... ……. 7
1.4.1.1 Bacterial conjunctivitis……………………………………....….. 7
1.4.1.2 Chlamydial conjunctivitis…………………………………....…… 8
1.4.1.3 Neonatal conjunctivitis (ophthalmia neonatorum)………….……. 9
1.4.1.4 Viral conjunctivitis……………………………………………… 9
1.4.1.5 Allergic (vernal) conjunctivitis………………………………….. 9
1.4.2 Eyelids Infections……………………………………………….. 10
1.4.2.1 Styes……………………………………………………….…….. 10
1.4.2.2 Chalazion………………………………………………….…….. 10
1.3.2.3 Blepharitis………………………………………………………… 11
1.4.3 Lacrimal apparatus infections………………………………..…… 11
1.4.3.1 Canaliculitis……………………………………………….……… 11
1.4.3.2 Dacryocystitis…………………………………………………… 11
1.4.4 Cornea infections (keratitis, corneal ulcers)………………. …….. 12
1.4.5 Orbital cellulitis……………………………………………..……. 13
1.4.6 Endophthalmitis…………………………………………..………. 13
1.5 Common bacteria associated with eye infection…………….…… 14
1.5.1 Gram-positive bacteria…………………………………………… 14
1.5.1.1 Staphylococci…………………………………………………… 14
1.5.1.2 Streptococci……………………………………………..……… 15
1.5.1.2.1 Streptococcus pyogenes………………………………………… 15
1.5.1.2.2 Streptococcus pneumoniae…………………………………..…… 16
1.5.1.3 Corynebacterium spp.…………………………………………… 17
1.5.1.4 Actinomyces spp…………………………………………..……… 18
7
1.5.1.5 Propionibacteria spp……………………………………………. 18
1.5.2 Gram-positive bacteria infrequently associated
With ocular infection………………………………………......... 19
1.5.3 Gram-negative bacteria………………………………………..….. 19
1.5.3.1 Neisseria………………………………………………….…….. 19
1.5.3.2 Moraxella spp…………………………………………….....……. 20
1.5.3.3 Pseudomonas spp…………………………………………….…. 21
1.5.3.4 Enterobacteriaceae…………………………………………..……. 21
1.5.3.5 Haemophilus spp………………………………………….....…. 22
1.5.3.6 Chlamydia trachomatis……………………………………..…….. 23
1.5.3.7 Gram-negative bacteria infrequently associated with ocular
infection………………………………………………………… 23
1.6 Diagnostic approach to bacterial ocular infection……………..…. 24
1.6.1 Specimen collection………………………………………………. 24
1.6.2 Staining and culture…………………………………………….. 25
1.7 Antibiotics treatment……………………………………..…….. 25
Chapter two: Material and Methods……………………………………. 27
2.1 Collection of samples…………………………………...……… 27
2.2 Media used for isolation, identification and sensitivity test……… 27
2.2.1 Solid Media…………………………………………………..…… 27
2.2.1.1 Blood Agar………………………………………………...……… 27
2.2.1.2 Nutrient Agar……………………………………………………... 28
2.2.1.3 MacConkey's Agar Medium………………………………….…... 28
2.2.1.4 Aeusculin Agar………………………………………….……… 29
2.2.1.5 Urea Agar………………………………………………….……… 29
2.2.1.6 Diagnostic sensitivity test Agar (D.S.T.A)……………….. ……... 30
2.2.1.7 Simmon's citrate Agar………………………………………..…… 31
2.2.2 Semisolid Medium………………………………………...……… 31
2.2.2.1 Motility Medium…………………………………………..……… 31
2.2.2.2 Hugh and Leifson's (OF) Medium…………………………...…… 32
2.2.3 Liquid media……………………………………………………… 33
2.2.3.1 Nutrient broth……………………………………………...……… 33
2.2.3.2 Peptone water……………………………………………...……… 33
2.2.3.3 Peptone water sugars…………………………………………… 34
2.2.3.4 Nitrate broth……………………………………………….……… 34
2.2.3.5 Voges-Proskauer test media………………………………….….. 34
2.3 Reagents………………………………………………………… 35
2.3.1 Hydrogen peroxide……………………………………………… 35
2.3.2 Oxidase test reagent…………………………………………..… 35
2.3.3 Nitrate test reagents…………………………………………..… 35
2.3.4 Voges-Proskauer test reagents………………………………..… 35
2.3.5 Kovacs' reagent………………………………………….……… 36
2.4 Indicators……………………………………………………….. 36
8
2.4.1 Bromothymol blue…………………………………………….….. 36
2.4.2 Andrade's indicator…………………………………………....... 36
2.5 Sterilization……………………………………………………… 37
2.5.1 Sterilization of glassware…………………………………..…… 37
2.5.2 Sterilization of media…………………………………………… 37
2.6 Preservation of media…………………………………………… 37
2.7 Isolation and Identification Methods………………………….… 37
2.7.1 Cultural methods………………………………………...……… 37
2.7.2 Primary identification test……………………………………… 38
2.7.2.1 Gram stain……………………………………………….……… 38
2.7.2.2 Morphological study…………………………………….……… 38
2.7.2.3 Oxidase test……………………………………………...……… 38
2.7.2.4 Catalase test……………………………………………..………. 39
2.7.2.5 Motility test……………………………………………..………. 39
2.7.2.6 The oxidation-fermentation test (OF) ………………………..… 39
2.7.2.7 Durham tube sugar fermentations ……………………………… 40
2.7.3 Secondary identification test……………………………………… 40
2.7.3.1 Coagulase test…………………………………………………… 40
2.7.3.2 Urease test……………………………………………….……… 41
2.7.3.1 Aesculin hydrolysis test ………………………………………… 41
2.7.3.2 Citrate utilization ………………………………………..……… 41
2.7.3.3 Voges-Proskauer (V-P)reaction………………………………… 41
2.7.3.4 Bile solubility…………………………………………………… 42
2.7.3.5 Nitrate reduction test…………………………………….……… 42
2.7.3.6 Novobiocin sensitivity test……………………………………… 42
2.8 Sensitivity test…………………………………………...……… 43
Chapter three: Results…………………………………………….……… 45
3.1 Gram-positive bacteria…………………………………..……… 45
3.1.1 Staphylococcus species………………………………….……… 45
3.1.2 Streptococcus species…………………………………………… 46
3.1.3 Corynebacteria species…………………………………..……… 46
3.2 Gram-negative bacteria………………………………….……… 47
3.2.1 Branhamella catarrahalis………………………………………. 47
3.2.2 Pseudomonas species…………………………………………… 47
3.2.3 Klebsiella pneumoniae ………………………………….……… 48
3.3 Antimicrobial sensitivity test in vitro…………………………… 48
Chapter four: Discussion…………………………………………..……… 64
Conclusion……………………………………………….……… 70
Recommendations……………………………………………….. 70
References……………………………………………………….. 72

9
List of figures

Supject Page
Fig (1) Eye swab culture grown on blood agar, showed large
beta-haemolytic colonies of Staphylococcus aureus…………… 59

Fig (2) Staphylococcus simulans on blood agar......................................... 60

Fig (3) Culture of streptococcus pneumoniae on blood agar
showed alpha-haemolytic colonies…………………………….. . 61

Fig (4) Pseudomonas aeruginosa on nutrient agar……………………... 62

Fig (5) Antibiotic sensitivity test of a bacterium isolated from
eye infection….............................................................................. 63

10