Study the Effect of Antibiotics and Propolis on Pathogenicity of The Methicillin Resistant ...

181 Pages·2014·1.7 MB·English

Republic of Iraq
Ministry of Higher Education
and Scientific Research
Baghdad University
College of Science

Study the Effect of Antibiotics and Propolis
on Pathogenicity of The Methicillin
Resistant Staphylococcus aureus (MRSA)

A Thesis
Submitted to the College of Science /University of
Baghdad in Partial Fulfillment of the Requirements for
the Degree of Master of Science in
Biology/Microbiology

By
Elaf Basim Nori AL-Sheikh
B.Sc. in Biology / University of Baghdad /College of Science
(2011)

Supervised by:
Assist.prof.Dr.Hanaa Saleem Yossef

Mahram /1435 November /2013
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[ ﺏِ ﺎَﺒﻟْ َﻷْ ﺍ ﻮُﻟﻭُﺃ ﻻﱠ ِﺇ ﺮُ ﻛﱠ ﺬﱠ َﻳ

ﻠﻴﻈﻌﻟﺍ ﺍ ﻠﻪ

(۲٦۹) ﺔﻳﻵﺍ / ﺓﺮﻘﺒﻟﺍ ﺓﺭﻮﺳ
Supervisor Certification

I certify that this thesis was prepared under my supervision at the
Department of Biology / College of Science / University of Baghdad, as
a partial requirement for the degree of Master of Science in
Microbiology.

Signature:
Asst.prof. Dr.Hanaa S. Yossef
Supervisor
Biology Department
College of Science
University of Baghdad
Date:

In view of the available recommendation, I forward this thesis for
debate by the examining committee.

Signature:
prof. Dr.Sabah N.Alwachi
Head of Biology Department
College of Science
University of Baghdad
Date:
Committee Certification
We, the examining committee, certify that we have read this thesis
and examined the student in its contents and that in our opinion; it is
adequate for awarding Degree of Master of Science in Microbiology.

Dr. Rajwa H.Essa
Chairman (Assistant Professor)
/ /2013

Dr. Sameer Abdul Ameer Abid Ali Alash Dr. Rana Saadi Aboud
Member (Lecturer) Member (Assistant Professor)
/ /2013 / /2013

Dr. Hanaa Saleem yossef
Supervisor (Assistant professor)
/ /2011

Approved by The Council of The College of Science, University of
Baghdad.

Prof.Dr. Saleh M. Ali
(Professor)
Dean of College of Science
University of Baghdad
/ /2013
declaration

This is to certify that the Desertation entitled:
“Study the Effect of Antibiotics and Propolis on Pathogenicity of
The Methicillin Resistant Staphylococcus aureus (MRSA)”.

Submitted by:
elaf basim nori al-sheikh
Department of: Biology
College of: Science
has been linguistically corrected and its language in
its
present form is acceptable.

dr. nazar aziz auda
Department of Biology,
College of Science, University of Baghdad
2013
Dedication

To

Whom who gave me their endless love,

Whom gave me their patience and support,

Who lighten my way to achieve this work,

I introduce my work with respect.

@
Elaf
Acknowledgements
.
Thanks for Al-mighty God for his generosity and mercy I would like to sincerely
thank and express of my great appreciation, heart felt gratitude and thankful to my
supervisor “Dr.Hanaa saleem ” for his scientific guidance, recommendation, advice,
encouragement and support, also I am grateful for his patience, generosity and
kindness during the all period of this research.
I would like to express of my profound thanks to the all staff members Department of
Biology, for their kindness, help and encouragement.
I express of my special thanks to the all members and staff in the Bacteriology Lab.
of AL-Kindy Teaching Hospitals, for their help in the collection of clinical
specimens and help in the identification of isolates.
Also I would like to express of deeply thanks and grateful to my friend “Dr. Thura
awad ” for her helpful, generosity and kindness.
My special, great and deepest love, thankful and grateful go to the most persons that
I love, whom without their patience, understanding, support and most of all love, the
completion of this work would not have been possible, “my lovely Family”.
Finally, I would like to thank everyone that helps me in some way or another during
my research work.
@
Elaf
summary
This study included collection of 100 swab specimens from
patients in AL-Kindy Teaching Hospital and teaching laboratories of
Medical City Hospitals in Baghdad during the period from August to
December 2012 , these swab specimens differed in their sources which
included 19 nasal swab, 16 wound swab, 27 burn swab, 7 pus swab, 15
sputum swab, 10 corneal swab and 6 urine swab . Only 38 (38%) isolates
were identified as Staphylococcus aureus, 38 isolates of them (82.6%)
were coagulase-positive (COPS), while 8 isolates(17.3%) were coagulase
negative (CONS), from total 46 isolates of Staphylococci.
The distribution of Methicillin resistance among Staphylococcus
spp isolates was investigated by disc diffusion method. In this study, 21
isolates (55.26%) out of 38 (38%) isolates were identified as
Staphylococcus showed to be resistant to the Methicillin while 17 isolates
(44.73%) were sensitive. The highest rate of Methicillin resistance
Staphylococci were obtained from wound and pus swabs.
The results obtained from antimicrobial sensitivity test showed that
MRSA isolates were resistant to many other antimicrobial agents in
addition to the Methicillin (Multi-drug Resistant).
Antimicrobial activities of different antimicrobial agents include
(crude Ethanolic Extract Propolis (EEP)), Lysostaphin, Ciprofloxacin and
Vancomycin) were tested singly and in combination against selected
isolates (MRSA S3) by using agar disc diffusion assay. Results revealed
that S. aureus (MRSA- S3) was more sensitive to 2 µg/ml concentration
of EEP than other antimicrobial agents used in this study.
However, antibacterial activity of the other three reagents were
significantly less than propolis, where MRSA isolate showed resistance
for their low concentrations. In comparing the pharmacodynamics of
lysostaphin, vancomycin and Ciprofloxacilin against selected isolate
(MRSA -S3) there was no significant differences with (P>0.05) between
those drugs at the diameter of inhibitions zone which were (11, 11and
10mm) respectively, the effected concentration of them were
(5.625µg/ml,16µg/ml and 3%).
The results showed a synergistic effect of this combination on the
selected isolate (MRSA- S3), as well as, same results obtained when
combined propolis with ciprofloxacin. The bacterial growth was
inhibited with elevation of inhibition zone to (11, 13mm) respectively,
while selected isolate (MRSA-S3) showed ahigh resistance against the
combination of vancomycin with (propolis and lysostaphin ) ,so they
effects was antagonistic to S. aureus.
The ability to produce slime layer by MRSA isolates was also
investigated and the results showed that all isolates of MRSA have
produced a slime layer when tested by tube method, but the amounts of
adherent materials were differ among the isolates. However, the results
by Congo red agar method showed that 57% of MRSA isolates produced
strong slime layer and 43% of MRSA revealed negative result. Similarly
the ability of MRSA to produce biofilm by tissue culture plate (TCP) was
investigated and the results indicated that MRSA isolates showed strong
ability to form abiofilm , and the OD value of biofilm formation ranged
between (0.262 - 0.311nm). moreover the OD value of biofilm formation
significantly increased after addition of 1% glucose to the media.
The Antimicrobial activity of propolis and lysostaphin on the
biofilm formed by MRSA isolates were investigated and the results
showed that propolis at MIC (2µg/ml) were significantly inhibited the
biofilm formation by MRSA S3 isolates when used alone, the optical
density significantly (P<0.05) decreased to (0.027nm) in comparison with
control group (0.3875nm).
Statistical analysis showed a slightly effect of lysostaphin under
therapeutic concentration (5.625µg/ml ) on biofilm formation ability of
(MRSA- S3) optical density was reduced to (0.312 nm) in compared to
control group (0.389nm). On the other hand, the results showed that the
synergistic combination of propolis with (lysostaphin, ciprofloxacin) at
therapeutic concentration were inhibited the biofilm formation by
selected isolates.
Histo-pathological effect of different concentrations of propolis,
lysostaphin and ciprofloxacin on the keratitis treatment induced in the
rabbit eyes was studied. Organs of rabbits (eyes) showed different
pathological changes in a corneas treated with 2µg/ml propolis alone
were significantly less inflamed than untreated MRSA S3–infected
corneas. The corneal epithelium and endothelium looked like an
uninfected cornea, clinical signs of keratitis in the experimental rabbit
were completely disappeared with score 0: they had normal and healthy
appearance compared with negative control group (uninfected eyes).
Corneas treated with lysostaphin were highly edematous in
comparison to uninfected eyes. Histopathological changes showed highly
neutrophils infiltrations with highly damaged endothelial layer, corneas
treated with lysostaphin had similar appearance to untreated corneas.

On the other hand, when combined propolis with lysostaphin and
propolis with ciprofloxacin results induced amoderate recovering in the
infected eyes, the pathological changes included moderate corneal
ulceration, conjunctively edema, and minor accumulation of fibrin in the
anterior chamber were the primary changes noted in those treated groups,
than untreated corneas. While when compared the current results with a
healthy cornea, it was found that the infected group still inflamed after