الفهرس 
LITERATURE REVIEWOnly 14 pages are availabe for public view
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Abstract
In most hospitals, patients’ food is prepared in the hospital kitchen and is then
distributed throughout the wards. Patients are more prone to infections in comparison to
healthy individuals. Even duration of hospitalization can be influenced by factors such as
malnutrition, food toxicities, and hospital infections. Thus, outbreaks of microorganisms in
hospitals can result in more serious outcomes for patients. Therefore, one of the principal
objectives of hospitals is provision of safe food for patients who are susceptible to
infections and complications.
Biofilms have been of considerable interest in the context of food hygiene. Of special
significance is the ability of microorganisms to attach and grow on food and food contact
surfaces under unfavorable conditions. Biofilm formation is a dynamic process and
different mechanisms are involved in their attachment and growth. Extracellular polymeric
substances play an important role in the attachment and colonization of microorganisms to
food contact surfaces. Various techniques have been adopted for the proper study and
understanding of biofilm attachment and control. If the microorganisms from food contact
surfaces are not completely removed, they may lead to biofilm formation and also increase
the biotransfer potential. Therefore, various preventive and control strategies like hygienic
kitchen layout and design of equipment, choice of materials, correct use and selection of
detergents and disinfectants coupled with physical methods can be suitably applied for
controlling biofilm formation on food contact surfaces.
The aim of this study was to evaluate the conditions of food contact surfaces, utensils
and equipment and the methods used to clean and sanitize them, in addition to the general
sanitary conditions of the kitchens and the personal hygiene in three chosen governmental
hospitals kitchens. Swabs from different food contact surfaces were collected from the
three kitchens and were subjected to microbiological examination for the detection of
E.coli, Salmonella and Staphylococcus aureus. Results of the biochemical examination of
swabs were confirmed using VITEK 2 COMPACT device. Then, isolates were tested for
their ability to form biofilm using the crystal violet binding assay on three surfaces (wood,
plastic and stainless steel) at two incubation periods (24 hr and 72 hr). Finally, isolates that
produced the highest amounts of biofilm on two or more surfaces were chosen to be
sequenced. One isolate from each type of microorganisms was chosen to be sequenced
using a sequencer.
I- Evaluation of the hygienic conditions of different food contact
surfaces commonly used in hospitals kitchens
Evaluation of the general hygienic conditions of the kitchens, the conditions of their
food contact surfaces, utensils and equipment and the methods used to clean and sanitize
them in addition to the personal hygiene of food handlers; were done using a pre-designed
checklist. It was obvious that the general hygienic conditions and the design of the three
kitchens were unsatisfactory. Sources of ventilation and light were inadequate. stainless
steel and marble are the materials from which worktops are made in the three kitchens.
Plastic cutting boards were used and they were in bad conditions, deeply stained and
scratched. No wooden food contact surfaces or cutting boards were used. No mechanical
dish washing machines were found. Incorrect methods for cleaning and sanitization were
used. Generally, sanitization process was not done either because of the absence of hot
water and/or the absence of approved food graded chemical sanitizer or due to the
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unawareness of the importance of this step. Cross contamination was avoided, through
using separate utensils and cutting boards for cooked and raw foods. Cleaning cloth were
not clean and were not cleaned properly between uses. The general personal hygiene and
the habits of the food handelers was unsatisfictory in the three kitchens. The total score
percentage for kitchen A was 49% , kitchen B was 34.7% and kitchen C was 49%. These
scores were unsatisfactory and indicating the presence of a severe hygienic problems.
II- Detection and isolation of some pathogens on food contact surfaces
used in the visited hospitals kitchens
• A total of ١٣٥ swabs were collected from the three kitchens, 45 swabs from each
kitchen and divided as follow (15 from stainless steel workbenches, 15 from
marble workbenches and 15 from plastic cutting boards.
• Microbiological examination for each sample was done for the detection of
E.coli, Salmonella and Staphylococcus aureus ,confirmation for the isolated
strains was done biochemically in our laboratories and, by using VITEK 2
COMPACT device.
• A total of 23 strains were isolated (11 isolates of Salmonella, 6 isolates of E.coli
and 6 isolates of Staphylococcus aureus). Also, 3 isolates of staphylococcus
epidermidis, 2 isolates of staphylococcus haemolyticus and one isolate of
pseudomonas aeruginosa were isolated.
• from kitchen A, one isolate of E.coli, two isolates of Salmonella, two isolates of
S.aureus, one isolate of S. epidermidis and one isolate of Pseudomonas
aeruginosa were isolated. In kitchen B, four isolates of E.coli, four isolates of
Salmonella,four isolates of S.aureus and two isolates of S. epidemidis were
isolated.While in kitchen C, five isolates of Salmonella, one isolate of E.coli and
two isolates of S. haemolyticus were isolated.
• Stock cultures of the purely identified isolates were maintained at 4°C on
Trypticase soy agar slant and refreshed monthly by subculturing. The purity of
each isolate was checked by streaking on blood agar or trypticase soy agar.
III- Testing isolated pathogens on different food contact surfaces
namely; plastic, wood and stainless steel were assessed for their
attachment and biofilm formation capability through using the
crystal violet binding assay
• Each microorganism was subcultured by streaking a loopful from its stock slant
culture onto the surface of Trypticase soy agar for isolation. Then one colony of
each microorganism was picked up by means of a sterile loop.
• Two hundred and seventy- six coupons (92 each (4 x 2 cm x 1.2 mm) for
stainless steel, wood and plastic were used. The coupons were washed with
detergent, and then rinsed 3 times in sterile distilled water, and air dried. The
coupons were wrapped in aluminum foil and placed in autoclave for sterilization
before use.
• A discrete colony of a 24 h test cultures on trypticase soy agar was used for
adherence assessment. One colony of each isolate was transferred into each of
sterile glass jars with lids containing 150 mL of trypticase soy broth. An
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uninoculated trypticase soy broth was used as a control. The inoculated broth
culture was incubated at 37°C for 24 h. After 24 h of incubation of inoculated
broth culture, four of steel, wood and plastic chips were put into each glass jar
with lid. All the glass jars were incubated at ambient temperature (26- 28°C) for
24 and 72 h. At the end of each incubation period, a set of chips (2 of each wood,
steel and plastic) were removed from broth culture for bacteria adherence
quantification.
• Detection of biofilm formation can be conducted in various ways, but crystal
violet assay was considered to be the most convenient technique to evaluate
bacterial adhesion and was done in this study. It involves washing, staining and
destaining of sessile cells with crystal violet where the optical density of the
stained bacterial biofilm was then determined spectrophotometrically. This
biofilm assay using crystal violet has been used to study biofilm formation by a
variety of Gram-positive and Gram-negative bacteria where it yields reproducible
result which allows one to study large numbers of strains and conditions at the
same time. Furthermore, the method yields quantitative results by measuring the
optical density.
• All the isolated E.coli strains produced biofilms on all types of the used surfaces
(wood, plastic and steel) at both incubation periods (24 and 72 h). where, the
average optical densities of all E.coli strains were, 1.276 on wood surfaces at 24 h
and 1.57 at 72 h incubation periods. And, 1.74 on plastic surfaces at 24 h and
increased to 1.902 after 72 h of incubation. While, on stainless steel was 0.636 at
24 h and reached 0.761 at the 72 h of incubation.
• Regarding Salmonella strains, the average optical densities of all strains were,
0.964 at 24 h of incubation on wood and increased to 1.059 at the 72 h of
incubation. while, on plastic were 1.232 at the 24 h of incubation and reached to
1.435 at 72 h of incubation. And on stainless steel, were 0.335 at 24 h and
increased to 0.426 at72 h.
• For S.aureus, the average optical densities on wood were 0.388 at 24 h and 0.46 at
the 72 h of incubation. And on plastic, were 0.688 at 24 h and reached 0.816 atthe
72 h. while, on stainless steel were 0.117 at the 24 h and 0.154 at 72 h of
incubation period.
• E.coli strains were found to produce the highest amounts of biofilm on all types of
the used surfaces (wood, plastic and steel) followed by Salmonella strains and the
least amounts of biofilm were produced by strains of S.aureus. Gram negative
organisms produced higher amounts of biofilms than gram positive organisms.
• Amounts of biofilm was the highest on plastic followed by wood surfaces which
are considered as hydrophobic materials, while the least amounts of biofilm were
found on stainless steel surfaces that is considered as hydrophilic material.
• Regardless the strain and the type of the surface, there was no significant
statistical difference in the amounts of biofilm that was produced at the 24 hr and
the 72 hr of incubation periods.
• The amount of biofilm to be produced will vary, depending on many factors such
as the type of the microorganism and even if the same organism there is individual
difference within the same species. Also, the type of the surface on which biofilm
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is formed. Moreover, the incubation period was not a factor that affects the
amounts of biofilm produced in our study.
• Then, one isolate from each type of microorganisms was chosen to be sequenced.
Isolates that produced the highest amounts of biofilm on two or more surfaces was
chosen to be sequenced. where, 16S rRNA analysis was made to obtain Pure PCR
products. The purified PCR products were sequenced by using 2 primers.
Sequencing were performed by using Big Dye terminator cycle sequencing kit.
Sequencing products were resolved on an Applied Biosystems model 3730XL
automated DNA sequencing system.
• Finally, it is recommended to clean the working surfaces in time before the
development of the biofilm, combine more than one cleaning method including
scrupping the surfaces to remove the formed biofilms and to select working
surface materials that do not support the growth of the biofilm.