الفهرس 
INTRODUCTION AND AIM OF THE STUDYOnly 14 pages are availabe for public view
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Abstract
Candida species are commensals of the human body which can become opportunistic
pathogens causing life threatening infections in immunocompromised patients. Candida
albicans is the most common species and it is responsible for about 90% of infections.
Several factors contribute to the virulence of Candida spp. such as adhesion, polymorphism,
extracellular enzyme production, and biofilm formation.
Various antifungal agents are used to treat Candida infections azoles, echinocandins,
polyenes, and nucleoside analogues. However, resistance to such agents has emerged which
necessitates studying other alternatives with lower resistance and fewer side effect. Garlic,
a well-known seasoning agent that has shown antifungal properties against Candida which
is mediated by interference with cellullar metabolism through inhibiting thiol-containing
amino acids and proteins.
Proteomics is a tool for analysis of proteins expressed by a given organism. It provides
a means for qualitative and quantitative analysis of proteins that interferes with cellular
biochemistry.
The present study aimed to investigate the antifungal activity of garlic extract and its
effect on the protein profile of Candida albicans.
The study was conducted at the Microbiology laboratory in the High Institute of Public
Health (HIPH) and two private microbiology laboratories over a period of 7 months, from
the 1st of January 2019 till the end of July 2019. It involved eighteen C. albicans isolates and
one ATCC (90028) C. albicans strain. The antifungal action of garlic on C. albicans
was evaluated in planktonic and biofilm states using broth microdilution test and
compared to fluconazole. Protein pattern of C. albicans was evaluated by sodium dodecyl
sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) after exposure to garlic extract and
fluconazole. SAP2 expression was assessed in 3 selected isolates after exposure to garlic
extract and fluconazole using Western blotting technique. Polymerase chain reaction (PCR)
and sequencing was performed to investigate the effect of garlic extract and fluconazole on
SAP2 gene.
The present study revealed the following results:
1- Seventeen isolates (94.4%) were sensitive to garlic extract while fourteen isolates
(77.7 %) were sensitive to fluconazole.
2- Garlic extract reduced the formation of biofilm in 72.2% and the maximum MBIC
among susceptible isolates was 4 times higher than MIC while 88.9% were inhibited
by fluconazole (n= 16) and the maximum MBIC was 2 times higher than MIC.
3- Garlic extract MBIC ranged from 0.87-112.4 mg/ml while fluconazole MBIC
ranged from 0.125-64 μg/ml.
4- Garlic extract eradicated the mature biofilm formed by 61.1% of isolates (n=11)
while fluconazole eradicated the mature biofilm in 88.9% of isolates (n=16).
5- Garlic extract MBEC ranged from 0.87- 224.8 mg/ml while fluconazole MBEC
ranged from 0.125-128 μg/ml.
6- Isolate 2 demonstrated a paradoxical growth of biofilm, after its inhibition with
garlic extract and fluconazole at concentrations exceeding MBIC.
7- Paradoxical growth or survivor cells appeared after exceeding MBEC of garlic
extract and fluconazole was observed in 7 and 9 isolates, respectively.
8- Exposure to garlic extract did not affect proteinase or phospholipase activity in
the clinical isolates (except for mild proteinase reduction in one clinical isolate)
or in the ATCC strain.
9- Exposure to fluconazole did not affect proteinase or phospholipase activity in
the clinical isolates (except for mild phospholipase reduction in one clinical
isolate) or in the ATCC strain.
10- Phospholipase and proteinase enzyme activity was not related to garlic extract
/fluconazole susceptibility.
11- Exposure to garlic extract, caused the number of protein bands to increase in all
clinical planktonic isolates (n=18) in addition to the ATCC strain with 13.4%
polymorphism.
12- Exposure to fluconazole caused an increase in the number of protein bands in 13
planktonic clinical isolates (72.2%) and also in the ATCC strain with 51.9%
polymorphism.
13- A dense band appeared approximately at 49-50 kDa in 88.9%, 72.2% of isolates
after garlic extract and fluconazole treatment of planktonic isolates, respectively
but it was denser in case fluconazole.
14- In garlic-treated biofilms, a band around 80 kDa appeared in 88.9% of biofilms
which was denser than in untreated biofilms.
15- In fluconazole-treated biofilms, a new band appeared at approximately 30 kDa in
88.9 % of fluconazole treated biofilms.
16- A new band at 60 kDa appeared in 94.4% and 83.3% of fluconazole treated and
garlic treated biofilms respectively.
17- Garlic extract produced more bands (median=16) than fluconazole (median =10) in
planktonic isolates while fluconazole produced higher polymorphism than garlic
extract (51.9% versus 13.4 %, respectively, p <0.001).
18- Garlic extract and fluconazole had a minimal effect on increasing the number of
bands in biofilms (median = 12 for both treated groups) and polymorphism was
higher in garlic-treated biofilms than in fluconazole treated ones (26% versus
21.8%, respectively, p=0.006).
19- The effect of garlic extract and fluconazole on protein band fragmentation in both
planktonic and biofilm forms was statistically significant in both treated groups
when compared to untreated isolates (p<0.001, p=0.006, respectively).
20- SAP2 expression levels were variable after treatment with fluconazole where the
highest level was (median =7.8 %).
21- Garlic extract caused a variation in SAP2 expression level where the highest was
(9.1%).
22- Based on sequencing data, the SAP2 sequence form the planktonic isolate treated
with garlic extract showed the highest dissimilarity from the reference sequence.
from the current study, it can be concluded that:
1- Garlic extract possesses antifungal, anti-biofilm properties against C. albicans.
2- Garlic extract had a better effect on planktonic cells while fluconazole had a better
effect on biofilm formation.
3- Garlic extract and fluconazole did not significantly affect phospholipase and
proteinase production in C. albicans.
4- Both garlic and fluconazole caused a change in protein fragmentation of C. albicans
in planktonic (with 13.4%, 51.9% polymorphism, respectively) and biofilm states
(with 26%, 21.8% polymorphism, respectively).
5- Exposure to garlic extract and fluconazole resulted in the appearance of new protein
bands in both planktonic and biofilm cells.
6- Exposure to garlic extract and fluconazole showed a high expression level of SAP2.
7- Exposure to garlic extract induced dissimilarity in the SAP2 planktonic sequence
compared with the reference sequence.
from the results of the present study, the following recommendations are
suggested:
1- The use of specific garlic isolated compounds rather than a whole garlic extract rather
to allow the identification of the exact garlic constituent causing antifungal activity
and affecting polymorphism in C. albicans.
2- Identifying polymorphic proteins which facilitates targeted- antifungal therapy.
3- Using a larger number of samples in Western blotting and sequencing, where specific
protein levels could be estimated and sequences could be identified