الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
 |  | from | 163 |  |  |
| | | from | 163 | | |
Abstract
The adhesion of pathogenic bacteria to epithelial cells is the first step towards development of an infection. This docking process is mediated by specific adhesions located on the outer microbial cell wall. Anti-adhesion therapy and anti-adhesion immunity are means to
reduce contact between host tissues and pathogens, either by prevention or reversal of adhesion of the infectious agent. It is well established that adhesion of enteric, oral and respiratory bacteria is required for colonization and for subsequent development of diseases.
The present study aims at isolation and collection of different Staphylococcal strains and evaluation of their abilities to adhere to eukaryotic Human Epithelial cells (HEp-2), subsequently different substances (plant extracts, organic and inorganic compounds) were assayed for their activities as anti-adhesion agents. The work in this study began with five bacterial strains; three standard Staphylococcal strains Staph. aureus ATCC 25923, Staph. aureus ATCC
6538 & Staph. aureus NCTC 6571 were obtained from American Culture Collection Institutes by El Nasr Pharma Company in Cairo; and two other bacterial isolates were isolated from twenty five different human infected tissues, from Tanta Cancer Center and the Faculty of Medicine Tanta University (El-Gharbia Governorate) These isolates were identified
through studying their morphological, physiological and biochemical
characteristics.
With concern to the best grown isolates we identified only two
isolates that were Staphylococcus epidermidis & Escherichia coli.
For obtaining the logarithmic-phase of the bacterial growth, bacteria
were grown for 3 h at 37°C, and measure turbidity every 30 min. by
spectrophotometer at 492 nm till it reach 0.5 and then harvested by
centrifuged and resuspended in RPMI 1640 culture medium. The bacterial
suspension was adjusted to a final concentration of 108 CFU/ml.
Human laryngeal Epithelial cells (HEp-2 cells) were prepared by
maintained in Dulbecco’s Modified Eagel Medium (DMEM) and incubated
in humidified atmosphere with 5% CO2.
Adhesion of bacterial strains on Human Epithelial cells (HEp-2) was
studied using [3- (4, 5- Dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium
bromide] MTT assay. Adhesion process was measured for the freshly
Summery - 97 -
prepared and overnight incubated culture and compared between them in
order to have the maximum adhesion for this study.
It was found that, the maximum value of the bacterial adherence was
recorded in the case of fresh cultures in Staph. aureus NCTC 6571(1.34)
followed by Staph. aureus ATCC 6538 and Staph. epidermidis (0.99)
followed by Staph. aureus ATCC 25923 (0.93), then E. coli (0.752) and
in overnight culture the maximum value of the bacterial adherence was
recorded in the case of Staph. aureus NCTC 6571 (1.8) followed by
Staph. epidermidis (0.85) followed by Staph. aureus ATCC 6538 (0.74)
then Staph. aureus ATCC 25923 and E. coli (0.66).
So the maximum value of the bacterial adherence was recorded in the
case of fresh culture in all strains while Staph. aureus NCTC 6571 only
showed more adherence in the case of overnight culture rather than fresh
culture.
Investigation of adhesion of Staphylococcus epidermidis by using
Scanning Electron Microscope. The adhesion and invasion of the strain
Staphylococcus epidermidis were detected by Scanning Electron
Microscope and anti-adhesion was also studied by the Scanning Electron
Microscope (SEM).
To explain the causes of bacterial adhesion (protein or
lipopolysaccarides), the bacterial strains under study were cultivated in
broth liquid medium for overnight and their lipopolysaccarides and
proteins were extracted and analyzed by SDS-PAGE Electrophoresis.
It was found that all tested bacterial strains that have different
adhesion values; have the same profile of protein and lipopolysaccarides
and this means that the main causes of the bacterial adhesion are neither
protein nor lipopolysaccarides but due to cell surface hyDROPhobicity.
Also the efficiency of different substances was determined as antiadhesion
agents for the pathogenic bacterial strains. By preparation of the
freshly prepared bacterial culture and they were mixed with different
substances (plant extracts, organic and inorganic compounds) separately to
measure their anti-adhesion activity by adding to HEp-2 cells.
Also activity of plant extracts was evaluated as anti-adhesion agents
by crude extracts of fresh leaves and aerial parts of three different plant
species. Nigella sativa, Trigonella fosnum and Eucalyptus globules were
assayed for their anti-adhesion activity.
It was found that the maximum anti-adhesion activity of the tested
plant extracts was recorded in the case of Nigella sativa extract followed
by the extract of Eucalyptus globules.
Summery - 98 -
Some organic compounds were also tested as anti-adhesion agents.
These were represented by different carbohydrate compounds including;
monosaccharides, disaccharides and polysaccharides.
It was found that all monosaccharide affect on adhesion of all strains.
The effect changed from strain to another one. The maximum effect on all
strains was with glucose except Staph. aureus ATCC 25923 which
recorded maximum effect with galactose in comparison with control which
contained no sugars.
Also disaccharide affect on adhesion of all strains. The effect
changed from strain to another one It was found the maximum effect on all
strains were with sucrose except Staph. aureus ATCC 25923 which
recorded maximum effect with lactose (0.26) in compared to control
(0.66).
In polysaccharide it was found that the maximum effect on all
strains E. coli (0.22), Staph. aureus ATCC 25923 (0.23) , Staph. aureus
ATCC 6538 and Staph. aureus NCTC 6571 (0.24) and Staph. epidermidis
(0.35) was with starch which gave best result rather than cellulose in
compared to control.
To evaluate the inorganic compounds as anti-adhesion agents,
different inorganic compounds including (NaCl & CaCl2) were tested for
their activity as anti-adhesion agents. It was found that the maximum antiadhesion
of the tested inorganic compounds was recorded in CaCl2 with
Staph. aureus ATCC 25923 and Staph. aureus ATCC 6538 (0.22) ,
Staph. aureus NCTC 6571(0.24) and Staph. epidermidis (0.28) while E.
coli was found to give the maximum result with NaCl (0.24) on HEp-2
cells compared to control.
So by studying all organic, inorganic and plant extract; it was
found that the best compounds were starch and CaCl2 for the purpose of
detection of the best concentration of this substances so different
concentrations (0.5, 1, 1.5 and 2 %) of the two substances were tested for
their activity as anti-adhesion agents and we found that; by increasing
concentration the anti-adhesion capacity increase.