1. health and fitness
2. disease

Republic of Iraq
Ministry of Higher Education
And Scientific Research
University of Baghdad
College of Science
Biotechnology Department
Biochemical and Molecular Study of
Staphyloxanthin Extracted from Clinical Isolates of
Staphylococcus aureus
A thesis
Submitted to the College of Science – University of Baghdad
As a Partial Fulfillment of the Requirements for the Degree of
Master of Science in Biotechnology
By
Eman Jihad Nafe'a AL-Kazaz
B.Sc., Biotechnology / College of Science
University of Baghdad (2006)
Supervised by
Prof. Dr. Alice K. Melconian
2014 A. C
January
Assist Prof. Dr. Nuha J. Kandela
1435 H
Rabe’a Al aw al
‫ٰ‬
‫ِ‬
‫ﺍﻟﺭ ﱠﺣ ْﻳ ِﻡ‬
‫ﻥ‬
‫ﻣ‬
‫ّ‬
‫ﻟﺭﺣ ّ‬
‫ﷲ ٓﺍ َٓ‬
‫ِﺑ ْﺳ ِﻡ َ‬
‫ﺐ ﻻَ ﻳَﻌﻠَ ُﻤ َﻬﺎ ﺇﻻﱠ ُﻫ َﻮ‬
‫َﻭ ِﻋﻨ َﺪﻩُ َﻣﻔَﺎﺗِ ُﺢ ﺍﻟ َﻐﻴ ِ‬
‫َﻭ ﻳَ َﻌﻠَ ُﻢ ﻣﺎ ﻓِﻲ ﺍﻟﺒَ ﱢﺮ َﻭ ﺍَﻟﺒَﺤ ِﺮ َﻭ َﻣﺎ‬
‫ﺗَﺴﻘُﻂُ ِﻣﻦ َﻭ َﺭﻗَ ٍﺔ ﺇﻻﱠ ﻳَﻌﻠَ ُﻤﻬﺎ َ َﻭ ﻻَ َﺣﺒﱠ ٍﻪ‬
‫ﺐ َﻭ ﻻَ‬
‫ﺕ ﺍﻷ ِﺭﺽ َﻭ ﻻَ َﺭﻁ ٍ‬
‫ﻓﻲ ﻅُﻠُﻤﺎ ِ‬
‫ﻴﻦ )‪(٥۹‬‬
‫ﺲ ﺇﻻﱢ ﻓﻲ ِﻛﺘَﺎ ٍ‬
‫ﺏ ُﻣ ِﺒ ٍ‬
‫ﻳَﺎﺑ ٍ‬
‫ﺻ َﺩ َﻕ ٰ‬
‫ّ‬
‫ﻅﻳﻡ‬
‫ﻌ‬
‫ﺍﻟ‬
‫ﷲ‬
‫ﱠ‬
‫َ‬
‫ِ‬
‫ﺳﻮﺭﺓ ﺍﻻﻧﻌﺎﻡ‬
Supervisor Certification
I certify that this thesis was prepared under my supervision in
Baghdad University-College of Science-Department of Biotechnology as
a partial requirement for the degree of Master of Science in
Biotechnology
Signature:
Signature:
Name: Dr. Alice Krikor Melconian
Name: Dr. Nuha Joseph Kandela
Title: Professor
Title: Assist Professor
Date:
/ / 2014
Date:
/ / 2014
In view of the available recommendations, I forward this thesis for
debate by the Examination Committee.
Signature:
Name: Dr. Abdul kareem Al-kazaz
Title: Chairman of Biotechnology
Date:
/ / 2014
Committee Certification
We, the examining committee, certify that we have read this thesis
and examined the student in its contents and that, according to our
opinion, is accepted as a thesis for the degree of Master of Science in
Biotechnology.
Signature:
Signature:
Signature:
Name: Dr. Abdul Kareem A. Al-kazaz
Name: Dr. Sawsan H. Othman
Name: Dr. Moruj A. al-obydi
Title : Assist Professor
Title: Assist Professor
Title : Assist Professor
Date:
/ / 2014
(Chairman)
Date:
/ / 2014
Date:
(Member)
/
/ 2014
(Member)
Signature:
Signature:
Name: Dr. Alice K. melconian
Name: Dr. Nuha J. Kandela
Title: Professor
Title: Assist Professor
Date:
Date:
/
/ 2014
(Member / Supervisor)
/
/ 2014
(Member/ Supervisor)
I hereby certify upon the decision of the examining committee
Signature:
Name: Salih Mehdi Ali
Title: Professor
Address: Dean of the College of Science
Date:
/ / 2014
Acknowledgments
In the name of ALLAH, most gracious, most merciful, praise to
ALLAH, the lord of the world and blessing be upon all his prophets
and upon the last prophet and messenger, Mohammed and upon his
family and his friends.
© I want to express my gratitude to my supervisors professor Dr.
Alice Krikor and Dr. Nuha Joseph, for their enthusiasm, their
inspiration and their great efforts to explain things clearly and
simply.
© A word of special thank is due to Dean of university and the
staff of Biotechnology Department at College of Science in
Baghdad university, head of Biotechnology Department Dr.
Abdul Kareem Al-Kazaz and to Dr. Ghazi Monem , Dr. Moruj
abdul Satar , Dr. Mohammed abdul Latif , Miss Rita Nabeel,
Miss Ramena Mekhael , Miss bayda'a Sezar, Miss Wasan
Wa'al and Mr. Laith .
© Iam especially grateful to Miss Nour Hashim for scientific
advice throughout my study in Biotechnology Research Center
of Al-Nahrain University.
© My thanks are also due to all staff involved in the field of work
at Bacteriology Department in centeric kids technical hospital
specially Mrs. Nesreen and Mrs. Asifa ali .
© Special thanks are extended to all friends specially Sara Kusai ,
Zainab Abas ,Zainab Anas and Dina Hamed .
© My deepest gratitude goes to my family ( my mother , my dear
husband for his assistance and encouragement throughout the
period of study, my brothers Ahmed and Ibrahim , my sisters
Rana and Fatin for their deepest love , support, encouragement
and helped me to have comfortable and smooth study
environment and special thanks to my sister Luma and all my
husband's relatives.
© Finally My great thanks to all which they helped me in anyway.
Eman AL-kazaz
Summary
I
Fourty three isolates ( 20.7%) characterized as Staphylococcus aureus ,
were isolated from 207 different clinical sources (blood , nose, , wound ,
urine , vaginal, ear and eye) in different percentages (30.23, 18.60, 16.28,
13.95, 15.15, 6.96 and 2.33) % respectively. The results showed that
blood samples were more accessible for contamination by this organism.
The antibiotic susceptibility test against 25 antibiotics revealed that all
isolates were
resistant
to Amoxicillin/Clavulanic acid, Aztreonam,
Carbenicillin, Ceftaxime, Cefotaxin, Cifixime, Nitrofluranton, Pencillin
G, Pipracillin and Vancomycin in 100%, whereas they indicated variable
resistance to the other antibiotics used. While Imipenem and
Trimethoprim were the most effective drugs used in the present study.
The results of staphyloxanthin production from S.aureus isolates on
different culture media ( Milk agar medium ,Trypticase yeast medium ,
Trypticase soya medium ,Peanut seed medium and
Sesame seed
medium) was 44.1%, 37.2%, 37.2%. 30.2 % , 30.2 % respectively
The Staphyloxanthin pigment was extracted using methanol and
purified partially by organic solvents and Thin Layer Chromatography
(TLC). The results revealed three peaks with a highest peak at (450) nm.
The local isolate Staphylococcus aureus AE36 was selected in this study
for its high productivity of Staphyloxanthin pigment (STX) .
The optimal conditions for pigment production by S. aureus AE36 ,
were detected and was noticed that the milk agar medium revealed the
highest production of pigment which was estimated 165.21unit/cell, at
pH 8 for 72 hr at 370C.
Summary
No antibacterial activity of STX was detected
used
in
this
study
(Staphylococcus
II
against the bacteria
epidermidis,
Pseudomonas
aeruginosa, Salmonella spp., Shigella spp., Escherichia coli, Klebsiella
spp , Proteus spp. Pseudomonas fluorescens , Pseudomonas putida ,
Staphylococcus aureus).
The wild-type (WT) S. aureus AE36 pigmented bacterium was mutant
using Ethyl Methane Sulphonate to inhibit the anti oxidant effect of the
pigment has impaired neutrophil survival and its impact as a virulence
factor , then compared with S.aureus AE38 the non pigmented bacterium
and the parent S. aureus AE36 , the results showed that the mutant wildtype S. aureus AE36 was less pathogenic in a mouse subcutaneous abscess
model and more susceptible to killing by neutrophil cell compared with
the parent wild-type S. aureus AE36.
Duplex real-time PCR assay represents a rapid and powerful method
used for the detection of crtM gene (staphyloxanthin pigment production)
and mecA gene (methicillin-resistance) in a single SYBR Green I RealTime PCR assay.
Introduction
۱
Staphylpococcus aureus is a major global public health problem
causing serious, often life threatening infections in the community and
hospital setting that are becoming more difficult to manage with current
antibiotics therapy regimens ( Brea et al .,2010). The pathogen
Staphylococcus aureus is a gram-positive, gold-colored bacterium of
which 20.8% is resistant to methicillin and all available β-lactam
antibiotics (Zetola et al .,2005). About 20% of the population is always
colonized with S. aureus, 60% are intermittent carriers, and 20% never
carry the organism (Peacock et al., 2001; VonEiff et al., 2001).
S. aureus expresses many potential virulence factors: surface proteins
that promote colonization of host tissues, Invasins that promote bacterial
spread in tissues (leukocidin, kinases, hyaluronidase), Surface factors that
inhibit phagocytic engulfment (capsule, Protein A), biochemical
properties that enhance their survival in phagocytes (carotenoids, catalase
production), Immunological disguises (Protein A, coagulase), membranedamaging toxins that lyse eucaryotic cell membranes (hemolysins,
leukotoxin, leukocidin), exotoxins that damage host tissues or otherwise
provoke symptoms of disease (Staphylococcal enterotoxin A - G, Toxic
Shock Syndrome Toxin, Exfoliative Toxin) and acquired resistance to
antimicrobial agents(Nester et al., 2001).
The yellow-to-orange colony color of S aureus is one of the classical
criteria for identification of this species. As early as 1882, Ogston
connected the yellow-orange appearance of pus with the color of the
infecting microorganisms (Kloos et al., 1991).
Later it was shown that the pigment should not be the only basis for
classification, since it is not a very stable character. Pigmentation is
۲
Introduction
usually apparent after 18 to 24 hr of growth at 37°C but is more
pronounced when cultures are held at room temperature for 24 to 48 h
longer.
Although loss of the ability to synthesize pigment in S aureus appears
to be irreversible, more than 90 % of strains isolated from clinical sources
are pigmented (Lennette et al.,1985). Staphyloxanthin is an membranebound carotenoid which plays a role in the environmental fitness of S.
aureus (Pelz et al., 2005 ;Clauditz et al.,2006). Membrane pigments have
also been hypothesized to be virulence factors in S. aureus, potentially by
detoxifying reactive oxygen species (ROS) produced by phagocytes (Liu
et al.,2008). Carotenoids may also stabilize the S. aureus membrane
during infection and pathogenesis (Rohmer et al.,1979).
The Molecular Weight of staphyloxanthin (STX) is 819.17 Daltons and
it is a typical secondary metabolite (Pelz et al., 2005). It is not necessary
for the growth and reproduction of S. aureus but might serve a role in
survival in infected hosts and in combating the immune system(Clauditz
et al.,2006). It has antioxidative functions that play role in light
harvesting,
energy
transfer,
and
the
regulation
of
membrane
fluidity(Albrecht et al., 2000 ; Holt et al.,2005 ). The protective functions
of carotenoids against oxidative stress, singlet oxygen, and peroxy
radicals promote the survival of pathogenic microbes during host immune
responses (Clauditz et al.,2006 ; Liu et al., 2008).
Staphyloxanthin is mainly producted in stationary phase, it scavenges
free radicals with its conjugated double bonds. Since staphyloxanthin is
located in the cell membrane, it probably protects lipids but might also be
involved in protecting proteins and DNA , and it plays an additional role
in the defense against damage by ROS, thereby enhancing the virulence
and fitness of the cells. Staphyloxanthin can be regarded as a biological
antioxidant against hydrogen peroxide and hydroxyl radicals and might
Introduction
۳
be useful as a therapeutic radical scavenger(Clauditz et al.,2006). The
caroteinoid biosynthesis genes were sequenced, and an operon containing
five genes (crtOPQMN) was identified, three further genes upstream of
crtM were found in addition to the known genes crtM and crtN, and they
found that all five genes are necessary for the formation of the orange
staphyloxanthin (Braun and Friedrich, 2001)
Consequently, the aim of the present study was to show the role of
staphyloxanthin pigment in S. aureus virulence and its ability to inhibit
other pathogenic bacteria.
Several investigations were carried out, to achieve the aims of the
present study. They are outlined in the following steps:
1-Isolation and identification of S .aureus from different clinical samples
and detecting the staphyloxanthin producer ones .
2- Determination the susceptibility of S. aureus isolates to different
locally used antimicrobials agents.
3--Extraction of staphyloxanthin pigment from the producer isolates and
determining its effect on other types of bacteria
4- Exposing the selected isolate to mutagenic agent (EMS), in order to
obtain a mutant that differs from the wild type.
5-Attampts to infect wounds in experimental animals with the bacteria
producing the pigment and comparing the results with the non producer
ones.
6- Detection of the Staphyloxanthin pigment and Methicillin-Resistant
Staphylococcus aureus (MRSA) isolates using SYBR Green RT-PCR
assay.
Chapter One
Literature review
4
literature review
1.1 General Description of Staphylococcaceae
This family of bacteria were 0.5–1.5 mm in diameter, occurring singly, in
pairs, in tetrads, in short chains (3-4 cells), and characteristically dividing in
more than one plane to form irregular grapelike clusters. Gram positive,
nonmotile, nonflagellate, nonspore-forming cell wall contains peptidoglycan
and teichoic acid. Usually unencapsulated or limited capsule formation.
Staphylococcacea are aerobic or facultative anaerobes grows well in medium
containing 10% NaCl, poorly in 15% NaCl ( William et al., 2009).
1.2 Classification of Staphylococcus aureus ( William et al., 2009).
Domain:
Bacteria
Kingdom:
Eubacteria
Phylum:
Firmicutes
Class:
Bacilli
Order:
Bacillales
Family:
Staphylococcaceae
Genus:
Staphylococcus
Species:
aureus
Chapter One
Literature review
5
1.3 General characteristics of S. aureus
No motile, non spore-forming, Gram positive cocci; occur singly and form
pairs and clusters. Facultative anaerobic. Grows' well in medium containing
10% NaCl, poorly in 15% NaCl. Positive reactions for alkaline phosphatase,
catalase, coagulase, heat-stable nuclease, hemolysis, and hyaluronidase.
Negative for oxidase, β-glucuronidase. Produces acid aerobically from fructose,
maltose, and sucrose. No acid production from arabinose, cellobiose,
melezitose, raffinose, salicin, xylitol, or xylose. Novobiocin-susceptible.
Peptidoglycan type l-Lys–Gly5–6. Teichoic acid contains ribitol and N
acetylglucosamine (Kloos and Bannerman, 1999). With two subspecies :
1. 3.1 Staphylococcus aureus subsp. aureus
Non motile, non spore-forming, Gram-positive cocci, 0.5–1.0 mm in
diameter; occur singly and form pairs and clusters. Colonies raised, smooth,
glistening, translucent, with entire margins. Pigmentation varies from gray to
yellow to orange. The pigments triterpenoid carotenoids and its derivatives of
them and are located in the cell membrane. Colony diameter > 5 mm . It may
produce capsules. Encapsulated strains usually produce smaller and more
convex colonies. Capsular polysaccharides
contain. N-acetyl-d-amino-
galacturonic acid, N-acetyl-d-fucosamine, and taurine. Cell membranes contain
glycolipids,
mono
and
diglucosyldiglyceride
phospholipids,
lysyl-
phosphatidylglycerol, and cardiolipin. Facultative anaerobic. Growth is best
under aerobic conditions. Temperature range from 10–45°C; optimum 30–
37°C. Growth good in medium containing 10% NaCl poor at 15% NaCl , (
Wilkinson et al .,1997 ;William et al., 2009).
Chapter One
Literature review
6
1.3.2 Staphylococcus aureus subsp. anaerobius
Nonmotile, nonspore-forming, Gram positive cocci, 0.8–1.0 mm in
diameter; occur singly and form pairs and clusters. Grows micro aerobically
and an aerobically . Produces l-lactate, acetate, and succinate an aerobically
from glucose. Colonies on blood agar are white, opaque, glistening, entire,
smooth, and convex. Colony diameter (1–3) mm after incubation for two days
on blood agar. Temperature range for optimal growth (30–40)°C. No growth at
20 or 45°C. All strains tolerate 10% NaCl; most do not tolerate 15% NaCl.
Distinguished from Staphylococcus aureus subsp. aureus by lack of pigment
and clumping factor and by the inability to carry out anaerobic fermentation of
mannitol, to grow at 45°C, to produce acetoin from glucose to reduce nitrate, to
produce b-glucosidase, and to produce acid from galactose, lactose, mannose,
mannitol, ribose, and trehalose ( William et al., 2009).
1.4 Pathogenicity
S. aureus is responsible for a variety of infections. In the late 1950s and
early 1960s, Staphylococcus aureus caused considerable morbidity and
mortality as a nosocomial pathogen of hospitalized patients. Among the major
human infections caused by this species are furuncles, carbuncles, impetigo,
toxic epidermal necrolysis (scalded skin syndrome)(Forbes et al.,2002),
pneumonia, osteomyelitis, acute endocarditis, myocarditis, pericarditis,
enterocolitis, mastitis, cystitis, prostatitis, cervicitis, cerebritis, meningitis,
bacteremia, toxic shock syndrome, and abscesses of the muscle, skin, urogenital
tract, central nervous system, and various intra-abdominal organs. In addition,
staphylococcal enterotoxin is involved in food poisoning (Stanier et al., 1986)
(Figure 1- 1). Methicillin-resistant Staphylococcus aureus (MRSA) strains have
Chapter One
Literature review
7
emerged in the 1980s as a major clinical and epidemiological problem in
hospitals. These strains are beginning to spread out of the hospitals and into
community Staphylococcus aureus is also capable of producing infections in a
variety of other mammals and birds.
Figure (1-1) Parts of the body and illnesses caused by S. aureus ( Todar, 2008).
1.5 Virulence factors of S. aureus
S. aureus is equipped with a wide arsenal of virulence mechanisms show in
(Table 1-1) and (Figure 1- 2). These are responsible for abscess formation,
evasion of host immune responses at many levels, and induction of the sepsis
syndrome (Raygada and Levine, 2009).
Chapter One
Literature review
Figure (1-2) S. aureus virulence factors (Olivier,2008)
8
Chapter One
Literature review
Table (1-1) Staphylococcus aureus virulence mechanisms (Teppo, 2012)
a
a: MSCRAMM, microbial surface components recognizing adhesive matrix molecules.
b: PVL, Panton-Valentine leucocidin
c: TSST, toxic shock syndrome toxin
d: PBP2a, an altered penicillin binding protein encoded by the mecA gene
e: VanA , a gene encoding vancomycin resistanc
9
Chapter One
Literature review
10
1.5.1 Enzymes
It produces various enzymes such as coagulase (bound and free coagulases)
which clots plasma and coats the bacterial cell which probably prevent
phagocytosis. Hyaluronidase also known as spreading factor that breakdown
hyaluronic acid and help in spreading of S. aureus. it also produces DNAse
(deoxyribonuclease) which breakdown the DNA ( Ziebandt et al.,2001).
1.5.2 Toxins
Depending on the strain, S. aureus is capable of secreting several exotoxins,
which can be categorized into three groups. Many of these toxins are associated
with specific diseases ( Jawetz et al., 1998).
1.5.2.1 Superantigens
Pyrogenic toxin superantigens (PTSAgs) have superantigen activities that induce
toxic shock syndrome (TSS). This group includes the toxin TSST-1, which causes
TSS associated with tampon use. This is characterized by fever, erythematous rash,
hypotension, shock, multiple organ failure and skin desquamation. Lack of
antibody to TSST-1 plays a part in the pathogenesis of toxic shock syndrome
(Forbes et al.,2002). Other strains of S. aureus can produce an enterotoxin that is
the causative agent of S. aureus gastroenteritis. This gastroenteritis is self-limiting,
characterized by vomiting and diarrhea one to six hours after ingestion of the toxin
with recovery in eight to 24 hours. Symptoms include nausea, vomiting, diarrhea,
and major abdominal pain( Harris et al.,2002).
Chapter One
Literature review
11
1.5.2.2 Exfoliative toxins
Exfoliative toxins are implicated in the disease of staphylococcal scaldedskin syndrome (SSSS), which occurs most commonly in infants and young
children. It also may occur as epidemics in hospital nurseries. The protease
activity of the exfoliative toxins causes peeling of the skin observed with SSSS
(Prevos et al., 2003).
1.5.2.3 Other toxins
Staphylococcal toxins that act on cell membranes include alpha toxin, beta
toxin, delta toxin, and several bicomponent toxins. The bicomponent toxin
Panton-Valentine leukocidin (PVL) is associated with severe necrotizing
pneumonia in children. The genes encoding the components of PVL are
encoded in bacteriophage found in community-associated methicillin-resistant
S. aureus (CA-MRSA) strains(Nester et al., 2001).
1.5.3 Other Immunoevasive strategies
1.5.3.1 Protein A
Protein A is anchored to staphylococcal peptidoglycan pentaglycine bridges
(chains of five glycine residues) by the transpeptidase sortase A(Schneewind et
al.,1995)Protein A, an IgG-binding protein, binds to the Fc region of an
antibody. In fact, studies involving mutation of genes coding for protein A
resulted in a lowered virulence of S. aureus as measured by survival in blood,
which has led to speculation that protein A-contributed virulence requires
binding of antibody Fc regions (Patel et al.,1987).
Chapter One
Literature review
12
Protein A in various recombinant forms has been used for decades to bind and
purify a wide range of antibodies by immunoaffinity chromatography (Wann et
al ., 1999). Transpeptidases, such as the sortases responsible for anchoring
factors like Protein A to the staphylococcal peptidoglycan, are being studied in
hopes of developing new antibiotics to target MRSA infections (Zhu and
Standland ,2008).
1.5.3.2 Staphylococcal Pigments ( Staphyloxanthin)
Some strains of S. aureus are capable of producing carotenoids named
staphyloxanthin, a golden colored carotenoid pigment. This pigment acts as a
virulence factor, primarily by acting as a bacterial antioxidant which helps the
microbe evade the reactive oxygen species which the host immune system uses
to kill pathogens(Liu and Nizet, 2009).
Mutant strains of S. aureus modified to lack staphyloxanthin are less likely to
survive incubation with an oxidizing chemical, such as hydrogen peroxide than
pigmented strains. Mutant colonies are quickly killed when exposed to human
neutrophils, while many of the pigmented colonies survive. In mice, the
pigmented strains cause lingering abscesses when inoculated into wounds,
whereas wounds infected with the unpigmented strains quickly heal(Clauditz et
al.,2006). These suggest that Staphylococcus strains use staphyloxanthin as a
defence against the normal human immune system. Drugs designed to inhibit
the production of staphyloxanthin may weaken the bacterium and renew its
susceptibility to antibiotics ( Liu et al.,2005).In fact, because of similarities in
the pathways for biosynthesis of staphyloxanthin and human cholesterol, a drug
developed in the context of cholesterol-lowering therapy was shown to block S.
Chapter One
Literature review
13
aureus pigmentation and disease progression in a mouse infection ( Liu et
al.,2008) .
The biosynthetic pathway of staphyloxanthin is shown in figure (1- 3).
Genetic analysis has revealed that the deletion of the early biosynthetic enzyme
dehydrosqualene synthase (CrtM) has no effect on the growth of S. aureus, but
results in marked attenuation of the virulence of S. aureus in a mouse model
(Pelz et al ., 2005 ) . Therefore, the staphyloxanthin biosynthetic pathway is a
potential target for the development of new anti-infectious agents against
MRSA by weakening its virulence.
The staphyloxanthin pigment is not completely secreted in media because a
fraction of it connected to the bacterial cell membarane ( Mishra et al ., 2009;
2011).
Figure (1-3) Proposed staphyloxanthin biosynthesis pathway ( Pelz et al., 2005)
Chapter One
Literature review
14
1.6 Antibiotic resistance of Staphylococcus aureus
Staphylococcal resistance to penicillin is mediated by penicillinase (a form of
β-lactamase) production: an enzyme that cleaves the β-lactam ring of the
penicillin molecule, rendering the antibiotic ineffective. Penicillinase-resistant
β-lactam antibiotics, such as methicillin, nafcillin, oxacillin, cloxacillin,
dicloxacillin, and flucloxacillin, are able to resist degradation by staphylococcal
penicillinase. Resistance to methicillin is mediated via the mec operon, part of
the staphylococcal cassette chromosome mec (SCCmec) (Berglund et al., 2009)
Resistance is conferred by the mecA gene, which codes for an altered
penicillin-binding protein (PBP2a or PBP2') that has a lower affinity for
binding β-lactams (penicillins, cephalosporins, and carbapenems). This allows
for resistance to all β-lactam antibiotics, and obviates their clinical use during
MRSA infections. As such, the glycopeptide vancomycin is often deployed
against MRSA (Choonkeun et al.,2012)
Aminoglycoside antibiotics, such as kanamycin, gentamicin, streptomycin,
etc., were once effective against staphylococcal infections until strains evolved
mechanisms to inhibit the aminoglycosides' action, which occurs via protonated
amine and/or hydroxyl interactions with the ribosomal RNA of the bacterial
30S ribosomal subunit (Carter et al.,2000) .There are three main mechanisms of
aminoglycoside resistance mechanisms which are currently and widely
accepted: aminoglycoside modifying enzymes, ribosomal mutations, and active
efflux of the drug out of the bacteria.
Aminoglycoside-modifying enzymes inactivate the aminoglycoside by
covalently attaching either a phosphate, nucleotide, or acetyl moiety to either
Chapter One
Literature review
15
the amine or the alcohol key functional group (or both groups) of the antibiotic.
This changes the charge or sterically hinders the antibiotic decreasing its
ribosomal binding affinity. In S. aureus, the best-characterized aminoglycosidemodifying enzyme is aminoglycoside adenyltransferase 4' IA (ANT(4') IA).
This enzyme has been solved by x-ray crystallography ( Sakon et al.,1993). The
enzyme is able to attach an adenyl moiety to the 4' hydroxyl group of many
aminoglycosides, including kamamycin and gentamicin.
Glycopeptide resistance is mediated by acquisition of the vanA gene. The
vanA gene originates from the enterococci and codes for an enzyme that
produces an alternative peptidoglycan to which vancomycin will not bind.
Despite this, MRSA generally remained an uncommon finding, even in
hospital settings, until the 1990s, when there was an explosion in MRSA
prevalence in hospitals, where it is endemic (Johnson et al.,2001)
MRSA infections in both the hospital and community setting are commonly
treated with non-β-lactam antibiotics, such as clindamycin (a lincosamine) and
co-trimoxazole (also commonly known as trimethoprim/sulfamethoxazole).
Resistance to these antibiotics has also led to the use of new, broad-spectrum
anti-Gram-positive antibiotics, such as linezolid, because of its availability as
an oral drug. First-line treatment for serious invasive infections due to MRSA is
currently glycopeptide antibiotics (vancomycin and teicoplanin). There are
number of problems with these antibiotics, such as the need for intravenous
administration (there is no oral preparation available), toxicity, and the need to
monitor drug levels regularly by blood tests. There are also concerns that
glycopeptide antibiotics do not penetrate very well into infected tissues (this is a
particular concern with infections of the brain and meninges and in
Chapter One
Literature review
16
endocarditis). Glycopeptides must not be used to treat methicillin-sensitive S.
aureus (MSSA) , as outcomes are inferior ( Blot et al.,2002).
Because of the high level of resistance to penicillins and because of the
potential for MRSA to develop resistance to vancomycin, the attending
physician may choose to use a glycopeptide antibiotic until the identity of the
infecting organism is known. After the infection is confirmed to be due to a
methicillin-susceptible strain of S. aureus, treatment can be changed to
flucloxacillin or even penicillin, as appropriate ( Sakon et al.,1993).
Vancomycin-resistant S. aureus (VRSA) is a strain of S. aureus that has
become resistant to the glycopeptides. The first case of vancomycinintermediate S. aureus (VISA) was reported in Japan in 1996 (Hiramatsu et
al.,1997), but the first case of S. aureus truly resistant to glycopeptide
antibiotics was only reported in 2002 ( Chang et al., 2003) .
MRSA is a highly adaptable organism. Its genome has evolved due to
mutation of its own genes and acquisition of exogenous genes (Otter and
French 2012). The ability of S. aureus to acquire antibiotic resistance
mechanisms has contributed to its emergence in both the community and
nosocomial settings (Zetola et al., 2005).
The methicillin resistance gene (mecA) encodes a methicillin-resistant
penicillin-binding protein (PBP2a) that is not present in methicillin-susceptible
S. aureus (MSSA) strains (Enright et al., 2002). mecA is carried on a mobile
genetic element called a staphylococcal cassette chromosome (SCC). SCCmec
can be thought of as an antibiotic 12 resistance island which carries the mec
gene complex that encodes β-lactam resistance and also contains transposons
Chapter One
Literature review
17
and copies of plasmids that carry various resistance genes against other non-βlactam antibiotics (Hiramatsu et al., 1997). At least five different SCCmec
forms have been described which differ in size and genetic composition and are
numbered from I to V (Zetola et al., 2005). Community-associated MRSA (CAMRSA) usually carries the type IV or V SCCmec. The smaller type IV cassette
usually only includes methicillin-resistance elements, which accounts for the
increased susceptibility to antibiotics other than β-lactams in the community
strains (Skov et al., 2012). The smaller size may also serve as an evolutionary
advantage by making type IV more amenable to horizontal spread (Zetola et al.,
2005). Antibiotic selective pressure is believed to be lower in the community
than in the hospital, hence the survival advantage of having multiple drugresistance is also lower (Chambers, 2001). Since there are generally less
antibiotics used in the community, community strains do not experience the
same selective pressure to carry resistance to multiple antibiotics as hospital
strains. There is no survival advantage in the community in carrying the larger
SCCmec cassettes. Although originally resistant only to β-lactams, many
MRSA strains have become resistant to multiple antimicrobials and are
currently usually susceptible only to glycopeptides such as vancomycin
(Enright et al., 2002).
Liu et al., (2008) observed that STX synthesis in staphylococcal cells is
associated with resistance to phagocyte-mediated killing in vitro and
staphylococcal persistence in target organs in relevant in vivo animal models. In
contrast, the potential role of S. aureus pigments in resistance to killing by non
oxidative host defenses has not been extensively studied. It is known that polar
carotenoids modulate the fluidity properties of natural and model lipid membranes
(Gruszecki and Strzalka,1991 ; Gruszecki, 1999; 2004), and such fluidity
Chapter One
Literature review
18
characteristics are critical to the interaction of membrane-targeting host defense
cationic antimicrobial peptides (CAPs) with S. aureus, the relationships among
STX production, cell membrane (CM) biophysics, and susceptibility to host
defense and other cationic peptides. The biosynthetic pathway for STX is shown in
figure (1-4). This figure includes those crt operon genes pivotally involved at the
various STX synthesis steps, as well as showing how the presence or absence of
carotenoids might theoretically impact staphylococcal membrane interactions with
cationic peptides ( Subczynski, et al 1992 ; Widomska,, et al 2006).
Figure (1-4) Biosynthetic pathway of carotenoid (STX) production and comparative model of
its effect on susceptibility versus resistance to host defense CAPs in S. aureus
(Mishra et al., 2011).
Chapter One
Literature review
19
1.7 Characteristic of Staphyloxanthin
The pigment name staphyloxanthin was first mentioned by Marshall and
Rodwell (1972).Staphyloxanthin (STX) is a membrane-bound carotenoid of
Staphylococcus aureus (Clauditz et al.,2006). It is well known that carotenoids
function as antioxidants, and it has been suggested that (STX) is a virulence
factor protecting Staphylococcus aureus
against oxidants produced by the
immunue system (Lang et al.,2000; Liu et al.,2005).
The pigment should not be the only basis for classification, since it is not a
very stable character. Pigmentation is usually apparent after 18 to 24 h of
growth at 37 0C but is more pronounced when cultures are held at room
temperature for 24 to 48 h longer. In particular, those S. aureus strains isolated
from multiply antibiotic resistant are yellow pigmented (Willis et al.,1964).
Nonpigmented (white) derivatives of S. aureus are often found in subcultures
of stored organisms (Pinner and Voldrich,1932;Doudoroff,1936). Although
pigment production is a rather unstable character, the possibility that the
respective genes are encoded on typical plasmids has been ruled out (Grinsted
and Lacey1973).
Although loss of the ability to synthesize pigment in S. aureus appears to be
irreversible, more than 90 % of strains isolated from clinical sources are
pigmented (Willis et al., 1966). Staphyloxanthin is responsible for its
characteristic golden colour of S. aureus . It is observed to be water soluble
pigment (Samaranika , 2012), and it is a typical secondary metabolite (Pelz et
al., 2005). it’s chemical formula(C51H78O8) (Figure1-5), and it’s molecular
weight (819.17) Daltons (Pelz et al.,2005). STX is a charge-neutral molecule
(Hartmann and Galla 1978 ; kim et al.,1991) and light had no effect on
Staphyloxanthin synthesis (Hammond and White 1970).
Chapter One
Literature review
20
Figure (1-5) Structure of Staphyloxanthin (Pelz et al.,2005)
1.8 Structure and Biosynthesis of Staphyloxanthin
The biosynthesis of the pigment starts with the head to head condensation of
two farnesyl diphosphate molecules, catalyzed by the dehydrosqualene synthase
CrtM, to yield 4,4'-diapophytoene (dehydrosqualene). Dehydrosqualene
desaturase, CrtN, catalyzes the formation of the first deep yellow-colored
carotenoid intermediate product, 4,4'-diaponeurosporene, which is formed via
successive dehydrogenation reactions (Wieland et al.,1994). The complete
staphyloxanthin biosynthesis operon is crtOPQMN and the genes of the crt
operon involved in staphyloxanthin biosynthesis .
The enzymatic reactions of staphyloxanthin biosynthesis pathway involved
five steps are :
· Condensation of two molecules of farnesyl diphosphate to form
dehydrosqualene catalyzed by the dehydrosqualene synthase CrtM.
· Stepwise oxidation of dehydrosqualene to 4,4'-diaponeurosporene catalyzed
by the dehydrosqualene desaturase CrtN.
Chapter One
Literature review
21
· Oxidation of the terminal methyl group of 4,4'-diaponeurosporene to form
4,4'-diaponeurosporenic acid, catalyzed by CrtP, which is probably a mixed
function oxidase.
· Esterification of glucose at the C1'' position with the carboxyl group of 4,4'diaponeurosporenic acid to yield glycosyl-4,4'-diaponeurosporenoate,
catalyzed by the glycosyltransferase CrtQ.
· Finally esterification of glucose at the C6'' position with the carboxyl group
of 12-methyltetradecanoic acid to yield staphyloxanthin, catalyzed by the
acyltransferase CrtO.( Marshall and Wilmoth 1981b) .
Marshall and Wilmoth (1981a) isolated the pigments of S. aureus S41 and
determined their chemical structure, identifying 17 compounds which are all
triterpenoid carotenoids possessing a C30 instead of the C40 carotenoid structure
found in most other organisms. The main pigment is staphyloxanthin, an Alpha Dglucopyranosyl-1-O-(4,4'-diaponeurosporene-4-oate)6-O-(12
methyltetradecanoate), in which glucose is esterified with both, triterpenoid
carotenoid carboxylic acid and a C15 fatty acid.
Ø
4,4'-Diapophytoene
Ø
4,4'-Diapophytofluene
Ø
4,4'-Diapo-7,8,11,12- tetrahydrolycopene
Ø
Neo-4,4'-diaponeurosporene C
Ø
4,4'-Diapo-ζ-carotene
Ø
Neo-4,4'-diaponeurosporene B
Ø
4,4'-Diaponeurosporene
Ø
4,4'-Diapolycopene
Ø
cis-4,4'-Diaponeurosporenal
Chapter One
Ø
4,4'-Diaponeurosporenal
Ø
4,4'-Diapolycopenal
Ø
4,4'-Diaponeurosporenol
Ø
cis-4,4'-Diaponeurosporenoic acid
Ø
4,4'-Diaponeurosporenoic acid
Ø
Isostaphyloxanthin
Ø
Staphyloxanthin
Ø
Glucosyl-diaponeurosporen- oate
Literature review
22
In S. aureus extract it was found that the concentration of staphyloxanthin
was only 50% of that of 4,4'-diaponeurosporene. This can be explained by the
observation that 4,4'-diaponeurosporene is already formed after 12 h
cultivation, while staphyloxanthin is produced later after 24 hr of incubation
Marshall and Wilmoth (1981a).
1.9 Optimization of conditions for staphyloxanthin production
1.9.1 Effect of medium composition
Bacterial production for secondary metabolite such as pigments and
antibiotics are effected by the nature of medium composition such as Yeast
extract (Xiong and Kapral ,1992), carbohydrate (Lactose , Dextrose )(
Hammond and White,1970) , trypticase ( Hammond and White,1970; Xiong
and Kapral ,1992) , sodium chloride (Wieland et al., 1994) and peptone (Xiong
and Kapral ,1992).All these compound are necessary for pigment
production.
Chapter One
Literature review
23
1.9.2 Effect of temperature
The temperature effects microorganisms growth and pigment production by
effecting the secondary metabolism in cell. The optimum temperature for
microorganism growth
doesn’t
correspond with temperature of pigment
production ,and it’s different in many organisms , for example S. aureus growth
temperature is between (10 – 37)oC ( William et al., 2009), whereas the
pigment production temperature is between (37 – 40) oC (Lacey et al ., 1970).
1.9.3 Effect of pH
The effect of pH in staphyloxanthin production is depend on the solubility of
medium composition , metabolism pathway and pigment production , therefore
the optimum pH for staphyloxanthin production is (7-8 ) (Xiong and Kapral
,1992 ; Wieland et al ., 1994) .
1.9.4 Effect of incubation time
Pigment production increased when the cell enters the stationary phase,
therefore the pigment production does not occur in lag phase but it start to
increase when the cell enters the log phase until it reaches its maximum at the
beginning of stationary phase after 72 hr of incubation (Clauditz et al., 2006)
1.9.5 Effect of aeration
The importance of aeration in staphyloxanthin production from S. aureus is
required soluble oxygen in the medium , different types of aerations can be
used: it can be by shaker incubator or by increase the surface area of media,
because the ratio of surface area to volume of media effect on pigment
production by oxygen pump that is necessary to pigment production , but the
Chapter One
Literature review
24
decrease of oxygen don’t effect on the growth of bacteria because the bacteria
facultatively anaerobic( Hammond and White ,1970; Resch et al.,2005 ;Wu et
al 2009).
1.10 Extraction and Purification of Staphyloxanthin
1.10.1 Extraction of Staphyloxanthin
Different methods of extraction the metabolic compound such as pigments
that producing from microorganisms depending on the nature of the compounds
or pigment and its characteristics and location for the cell, since some of the
products secreted outside of cells to the culture medium, and some of which
remain inside the cell. For extract those products must break the walls of cells
by two ways including physical methods such as ultrasound or using chemical
methods such as The use of organic solvents and alcohol that lead to melt fat
connected in the cell walls(Ahmad et al ., 2012).Whereas Anuradha et al.,2004
used ethyl acetate to extract staphyloxanthin ,Marshall and Wilmoth (1981a)
used methanol , Ra’oof and Latif ,2010 used Chloroform and Giri et al., 2004
used acetone for extract the pigment .
1.10.2 Thin-layer chromatography
Thin-layer chromatography (TLC) is used to separate mixtures of two or more
compounds by distribution between two phases, one of which is moving (the
solvent) and the other that is stationary (the solid media). Prepared plates are
made of a porous adsorbent solid media (silica gel) which is adhered to a thin
piece of glass or plastic. The chromatography process works by differences in
polarity of the compounds present. Polar compounds include carboxylic acids,
amines, alcohols, esters aldehydes and ketones. Less polar compounds include
Chapter One
Literature review
25
aromatic compounds, halocarbons, ethers, olefins and hydrocarbons. Organic
molecules will bind to fine particles of the silica gel by intermolecular forces.
· Non polar compounds such as aromatic compounds bind to the silica gel
via weak van der Waals forces.
· Polar compounds will bind to the silica gel more strongly via dipoledipole interactions, hydrogen bonding or salt formation.
TLC can be used to determine if a substance is pure (one spot appears on the
plate after developing) or a mixture (two or more spots on plate). When
separating a mixture, one chooses a solvent that will cause the compounds,
which hopefully all have different polarities to move across the TLC plate at
different rates(Meisen et al., 2011).
1.11 Genetic expression of staphyloxanthin
The gold color of S. aureus is derived from the yellow-orange carotenoid
staphyloxanthin,a
virulence
factor.
The
chemical
characterization
of
staphyloxanthin, combined with analysis of S. aureus mutants, enabled the
elucidation of the staphyloxanthin biosynthetic pathway(Marshall and
Wilmoth,1981b) which was thought to consist of
etal.,2005) :
Ø
4,4′-diapophytoene synthase(CrtM)
Ø
1, 4,4′-diapophytoene desaturase(CrtN),
Ø
4,4′-diaponeurosporene oxidase(CrtP),
Ø
glycosyltransferase(CrtQ),
Ø
acyl transferase(CrtO)
five enzymes (Pelz
Chapter One
Literature review
26
CrtP introduces a terminal oxygen molecule into 4,4’-diaponeurosporene,
which results from sequential activities of CrtM and CrtN, to form a carboxylic
acid intermediate (4,4’-diaponeurosporenoic acid) via an aldehyde intermediate.
Staphyloxanthin is finally synthesized by further modification of4,4′diaponeurosporenoic acid by glycosylation (CrtQ) and acylation (CrtO) at a
terminal carboxyl group. Interestingly, CrtP was reported to function as both an
oxidase and an aldehyde dehydrogenase (Mijts etal.,2004) .
1.12 The crt operon
The carotenoid biosynthesis genes on plasmid pOC1 were further sequenced,
and an operon containing five genes (crtOPQMN) was identified. In addition to
the known genes crtM and crtN three further genes upstream of crtM were
found, by analysis of deletion mutants, it turned out that all five genes are
necessary for the formation of the orange staphyloxanthin. The organization of
the operon is shown in figure (1-6).
Figure ( 1-6) Organization of the staphyloxanthin biosynthesis genes of S. aureus
( Braun and Friedrich ,2001)
Chapter One
Literature review
27
1.13 Mutation of Staphyloxanthin
The crtM gene in the chromosome of S. aureus was exchanged with the gene
encoding chloramphenicol transferase (cat) through a double-crossover event. The
resulting mutant, S. aureus ∆crtM did not produce C30-carotenoids and formed
colorless colonies on Trypticase soya agar ( TSA) plates. This loss of pigmentation
also showed that no alternative pigment biosynthesis pathway exists in this strain.
No differences between the growth of the wild type strain and of the ∆crtM mutant
were observed. The inserted cat gene, which lacks a promoter, was also used as a
reporter gene to study the regulation of the pigment biosynthesis genes in S. aureus
strain. In the ∆crtM mutant, the cat gene is under the control of the promoter of the
crt operon. Therefore, the transcriptional regulation of the crt operon was studied
by following Cat activity during the growth of the culture. The Cat activity
increased significantly at the beginning and during the stationary phase, which is
in agreement with earlier observations of a marked increase in the pigmentation of
the wild type strain only after 24 to 36 h of growth (Wieland et al .,1994).(Figure1-7).
1.14 Regulated promoter of the crt operon from S. aureus
The loss of the sigB gene and the regulatory genes rsbV and rsbW leads to a
loss of pigmentation in S. aureus. In addition, the colorless S. aureus carrying
plasmid pIK57 (a pTX15 derivative with an inducible sigB gene (Kullik et al
.,1998) is pigmented after sigB induction. Based on these results, a B-regulated
crt promoter has been hypothesized (Kullik and Giachino 1997;Kullik et al
.,1998). Further Characterization of the crt operon promoter region indicated
that indeed the crt operon promoter is regulated by sig B. The transcription start
point of the crt operon was identified using primer-extension analysis.
Chapter One
Literature review
28
Figure (1-7) crt operon and its expression . A, organization of the crt genes in the S. aureus
genome ,B, construction of truncated crt expression plasmids. C, colonies of non pigment
colony and pigmented colony expressing staphyloxanthin ( Pelz et al., 2005).
1.15 Real Time PCR technique
Multiplex and real-time PCR are molecular techniques designed to amplify
nucleic acid sequences in a quantitative manner .Real-time polymerase chain (RTPCR) reaction, also called quantitative real-time PCR (qRt-PCR) is used to amplify
and quantify targeted DNA molecules. The use of RT-PCR allows for both
detection and quantization of DNA sequences. The quantity can be an absolute
number of copies or a relative amount when normalized to DNA input or
additional normalizing genes. The procedure for RT-PCR follows the general
principles of PCR, but the defining feature is the ability to detect amplified DNA
as the reaction progresses in real time.
Chapter One
Literature review
29
Real-time PCR can used to amplify low-abundance DNA templates. It is useful
in monitoring the accumulating amplicon. Two common methods that are used to
product detection in real-time PCR include the use of non-specific flourescent dyes
that intercalate with double-stranded DNA or sequence-specific DNA probes that
consist of oligonucleotides labeled with a fluorescent reporter (oligoprobes). The
fluorescent reporter permits detection after hybridization of the probe with its
complementary DNA target (Wawrik et al ., 2002)
Multiplex-PCR consists of multiple primer sets within a single PCR mixture
to produce amplicons of varying sizes that are specific to different DNA
sequences. By targeting multiple genes at once, additional information may be
gained from a single test run that otherwise would require several times the
reagents and more time to perform. Annealing temperatures for each of the
primer sets must be optimized to work correctly within a single reaction, and
amplicon sizes, i.e., their base pair length, should be different enough to form
distinct bands when visualized by gel electrophoresis. Commercial multiplexing
kits for PCR are available and used by many forensic laboratories to amplify
degraded DNA samples.( Hayden et al ., 2008).
Chapter two
Materials and Methods 30
MATERIALS AND METHODS
2.1 Materials
2.1.1 Instruments and Equipments
All instruments and equipments used in this study are mentioned in table (2-1).
Table (2-1) Instruments and equipments used
Instrument or equipment
Company
Origin
Autoclave
RILP (TR280D)
Italy
Balance
Denver
Germany
Cold Microfuge
Eppendorf
Germany
Compound light Microscope
Novex
Holand
Cooling Centrifuge
Eppendorf
Germany
Deep Freezer
Sanyo
Japan
Electrophoresis Unit
LKB
Sweden
Gel Documentation System
Bionner
Korea
Incubator
Memmert
Germany
Laminar Air Flow Hood
Techne
UK
Magnetic Stirrer With Hot Plate
Scientific Industries
USA
Microfuge
Beckman
USA
Micropipette
Gilson
France
Microwave Oven
LG
Korea
Mill
Natinal
Japan
Millipore Filter Paper
Milliporecorp
USA
Chapter two
Materials and Methods 31
Mixer
Griffin
Germany
Oven
Heraens
Germany
pH Meter
Oakton
China
Power Supply
Consort
Belgium
Real-Time Thermocycler
Applied 7500
USA
Refrigerator
National
Japan
Sensitive Balance
Sartaurus
Germany
Shaking Incubator
Adolf Kuner
Switzerland
UV Spectrophotometer
Optima
Japan
Visible Spectrophotometer
Banch & Lomb
Germany
Water Distillator
G.F.L
Germany
instrument
2.1.2 Chemical and biological compounds
All materials and compounds used in this study are presented in table (2-2).
Table (2-2) Chemical and biological compounds used
Chemicals
Company
Origin
Absolute alcohol
BDH
England
Aceton
BDH
England
Agar-Agar
Himedia
India
Agarose
Promega
USA
BDH
England
Ammonium chloride (NH4CL)
Chapter two
Ammonium Dihydrogen Phosphate
Materials and Methods 32
Fluka
England
Amyl-alcohol
BDH
England
Arabinose
BDH
England
Barium Chloride (BaCl2.2H2O)
BDH
England
Boric acid
Fluka
Switzerland
Bromocresol Purple
BDH
England
Buffer solution for Gimsa stain
BDH
England
Chloroform
Park
UK
Crystal violet
Fluka
England
Dipotassium Phosphate (K2HPO4)
BDH
England
Ethanol 99%
BDH
England
Ethdium bromid
BDH
England
Ethyl acetate
BDH
England
Ethyl methan sulphonate(EMS)
Sigma
India
Ethylene Diamin Tetra Acetic acid
Scharlan-
Spain
(EDTA)
Barcelona
( NH4H2PO4)
Ethylene Diamine Tetraacetic Acid
LTD
England
BDH
England
Ficol
BDH
England
Gelatin
BDH
England
Disodium (Na2- EDTA)
Ferrous Ammonium Sulfate
(NH4)2Fe(SO4)2.6H2O
Chapter two
Materials and Methods 33
Glucose
BDH
England
Glycerol
Oxoid
England
Hydrochloric acid (HCL)
BDH
England
AL-Razi
Iraq
Iodine
BDH
England
Leishman Stain
BDH
England
Magnesium sulfate
Fluka
England
Mannitol
Fluka
Switzerland
Mannose
BDH
England
Methanol
Difco
England
Peptone
Oxoid
England
Potassium chloride
Sigma
India
Potassium dihydrogen phosphate
BDH
England
Potassium Hydroxide
Merck
England
Raffinose
Fluka
Switzerland
Safranine
Fluka
England
Skim Milk
Himedia
India
Sodium chloride (NaCL)
BDH
England
Sodium dodesyle- sulphat (SDS)
LTD
England
Sodium thiosulphate
BDH
England
Hydrogen peroxide (H2O2 6%)
heptahydrate(MgSO4.7H2O)
(KH2PO4)
Chapter two
Materials and Methods 34
Sucrose
BDH
England
Terato methyl-p-phenylene
Sigma
India
Toluidine Blue
Fluka
Switzerland
Trehalose
BDH
England
Tris-base
LTD
England
Trypton
Difco
England
Xylene
BDH
England
Xylose
BDH
England
Yeast extract
Himedia
India
α- Nephthol
BDH
England
diminedihydrochloride
2.1.3 Enzymes
Enzymes used throughout this study are listed in table (2-3)
Table (2-3) List of enzymes used in the study
Enzymes
Company
Origin
Lysozyme
BDH
England
Proteinase - K
BDH
England
2.1.4 Other Material
Different materials were used in this study are maintained in table (2-4)
Chapter two
Materials and Methods 35
Table (2-4) Other materials were used
Material
Company
Origin
Bio Merieux
France
crtM Primer
Bionner
Korea
crtN Primer
Bionner
Korea
Human Blood
Education Lab
Baghdad
Human Plasma
Education Lab
Baghdad
Peanut seeds
Local market
Iraq
Sesame seeds
Local market
Iraq
Merck
Germany
Local market
Iraq
Bionner
Korea
Api-20 Strip Staph
Silica gel chromotography
Sunflower seeds
SYBER Green Master mix
2.1.5 Culture Media
All Ready-made media used in this study were prepared according to the
manufacturer's company instructions: other culture media were prepared in the
laboratory according to scientific references. All these media except maintained
were sterilized by autoclave at 121C° for 15 minutes at 15 pound per squared
inch ( PSI ).
2.1.5.1 Ready-made media
The ready-made media used in this study are illustrated in table (2-5)
Chapter two
Materials and Methods 36
Table (2-5) List of the diagnostic media used.
Culture media
Company
Origin
Blood agar base
Himedia
India
Brain-Heart Infusion Agar
Himedia
India
Himedia
India
DNase
Himedia
India
MacConkey’s Agar (M.A)
Himedia
India
Mannitol salt agar (MSA)
Himedia
India
Mueller-Hinton
Himedia
India
Nutrient Agar (N.A)
Himedia
India
Nutrient Broth (N.B)
Himedia
India
Plate count agar
Oxoid
England
Himedia
India
Himedia
India
(BHIA)
Brain-Heart Infusion Broth
(BHIB)
Agar
(MHA)
Trypticase
Soya
Broth
(TSB)
Urease agar
2.1.5.2 Prepared culture media
All prepared media were sterilized by autoclaved at 1210C for 15 min at 15
pound per square inch.
Chapter two
Materials and Methods 37
2.1.5.2.1 Blood Agar
This medium was prepared by adding 7% (v/v) of sterile human blood to
blood agar base medium (section 2.1.5.1). It was used to cultivate bacterial
isolates and study the type of blood hemolysis (Collee et al., 1996).
2.1.5.2.2 Gelatin Agar Medium
It was prepared by adding 4.4% of gelatin to nutrient agar medium (section
2.1.5.1).The pH was adjusted to 7.2 and autoclaved. This medium was used for
detection of proteolytic activity or gelatin liquefaction (MacFaddin, 2000).
2.1.5.2.3 Deoxyribonuclease (DNase) media
It was prepared by adding 0.005gm of toluidine blue dye to sterilized DNAse
medium (section 2.1.5.1). This medium was used to detected the ability of
bacteria to degraded of nucleic acid ( Jeffries et al ., 1957).
2.1.5.2.4 Milk agar medium
It was prepared by adding of 15% skim milk to sterilized nutrient agar
medium (section 2.1.5.1) (Harrigan and MacCance, 1976).
2.1.5.2.5 Peanut seeds medium
It was prepared by adding 5% of milled peanut seeds (section 2.1.9.12) to
nutrient agar medium (section 2.1.5.1). This medium was used for the detection
of pigmentation ( Giri et al .,2004).
Chapter two
Materials and Methods 38
2.1.5.2.6 Sunflower seed medium
It was prepared by adding 5% of milled sunflower seeds (section 2.1.9.12) to
nutrient agar medium (section 2.1.5.1).This medium was used for the detection
of pigmentation (Giri et al .,2004).
2.1.5.2.7 Sesame seed medium
It was prepared by adding 5% of milled sesame seeds (section 2.1.9.12) to
nutrient agar medium (section 2.1.5.1). This medium was used for the detection
of pigmentation ( Giri et al .,2004).
2.1.5.2.8 Trypticase yeast medium
It was prepared by adding 1% of yeast extract , 1% agar-agar, 1.7gm Peptone,
0.5 % sodium chloride to triypticase soya broth (section 2.1.5.1) .This medium
was used for the detection of pigmentation (Xiong and Kapral , 1992).
2.1.5.2.9 Carotinoid Expression Medium
It was prepared by adding 0.4 % glycerol to trypticase yeast medium( section
2.1.5.2.8) . pH was adjusted to 7.9 . This medium was used for the detection of
pigmentation (Xiong and Kapral , 1992).
2.1.5.2.10 Trypticase soya medium
It was prepared by adding 1% agar-agar to triypticase soya broth prepared in
(section 2.1.5.1). This medium was used for the detection of pigmentation .
Chapter two
Materials and Methods 39
2.1.5.2.11 Carbohydrate Fermentation Medium
It was prepared by adding 0.2 gm yeast extract , 0.1gm NH4H2PO4 , 0.02gm
Potassium chloride , 0.02 gm MgSO4.7H2O , 0.004 gm Bromocresol purple ,
0.1gm agar- agar to 100 ml distilled water .The pH was adjusted to 7.4 and
sterilized by autoclaving at 1210C for 15 min at 15 pound per square inch. Then
the carbohydrates ( Sucrose , Arabinose , Melebioze , Raffinose , Mannose ,
Xylose , Trehalose ) were added individually after being sterilized using
Millipore filter (0.45µm ) to obtain final concentration 1 % ( Baird.,1996).
2.1.5.2.12 Milk broth medium
It was prepared by adding of 15% skim milk to sterilized nutrient broth
medium (section 2.1.5.1) , then cooled to 500C . This medium was used to
detected the ability of bacterial isolates that produce alkaline protease and
pigment (Harrigan and MacCance, 1976).
2.1.6 Analytical Profile Index ( API System)
API – Staph . Ident . System ( BioMerieux , France) was used to confirm the
biochemical identification of the isolated bacteria.
2.1.7 Antimicrobials
2.1.7.1 Antimicrobial Disks
The antimicrobial disks presented in table (2-6) were used for detecting the
susceptibility of S.aureus isolates to these antibacterials. The results of this
experiment were recorded according to the standard guidelines recommended by
National Committee for Clinical Laboratory Standards (NCCLS, 2007).
Chapter two
Materials and Methods 40
Table (2-6) the antimicrobial disks used in the study (NCCLS, 2007).
Antimicrobial Disks
Symbol
Concentration
µg/disk
Amikacin
AK
30
Amoxicillin/clavulan
ic acid
AC
20/10
A
10
Azithromycin
AT
15
Aztreonam
AO
30
Carbenicillin
CB
100
Ceftaxime
CE
30
Cefotaxin
CN
30
Ceftazidime
CA
30
Ceftriaxone
CI
30
CFX
5
Ciprofloxacin
CF
5
Erythromycin
E
15
Gentamicin
G
10
Imipenem
I
10
Levofloxacin
LE
5
Methicillin
M
5
Nitrofurantoin
NF
300
Oxacillin
OX
1
Pencillin G
P
10 unit
Pipracillin
PC
100
Rifampin
R
5
Tetracycline
T
30
Trimethoprim
TR
5
Vancomycin
VA
30
Ampicillin
Cifixime
Company
Origin
Himedia
India
Chapter two
Materials and Methods 41
2.1.7.2 Antimicrobial ointment
The antimicrobial ointment used in this study is presented in table (2-7).
Table (2-7) The antimicrobial ointment used
Antimicrobial
Symbol
Concentratio
Company
Origin
Samara
Iraq
n µg/ml
Ciprofloxacin
Cf
5
(ointment)
2.1.8 Bacterial Isolates
The bacterial isolates used in this study are shown in table (2-8).
Table (2–8 ) The bacterial isolates used throughout the study
Bacterial Isolate
source
Pseudomonas aeruginosa
Pseudomonas putida
Pseudomonas fluorescens
Staphylococcus epidermidis
Department
of
Biotechnology
University of Baghdad
Escherichia coli
Klebsiella spp.
Salmonella spp.
Shigella spp
Proteus spp.
/
Chapter two
Materials and Methods 42
2.1.9 Solutions and Reagents
2.1.9.1 Gram stain solutions
Gram stain including Crystal Violet, Iodine, Ethanol and Safranine ,stains were
provided by AlRazi Company / Iraq.
2.1.9.2 Oxidase Test Reagent
This reagent was prepared instantly by dissolving 1gm of tetramethylparaphenylnene-diamine-dihydrochloride in 100 ml distilled water and stored in
a dark bottle (Collee et al., 1996).
2.1.9.3 Catalase Reagent (3%)
It was prepared by adding 1ml of 6 % hydrogen peroxide solution to 1 ml of
distilled water (Cruikshank et al., 1976).
2.1.9.4 Normal saline solution
This solution was prepared by dissolving 8.5gm of NaCl in a 50 ml of distilled
water and then completed the volume to 100 ml distilled water, autoclaved at
121° C for 15minutes(Atlas et al., 1995)
2.1.9.5 Coagulase test
Fresh human plasma was used in this test (Baron et al., 1994a).
2.1.9.6 Stock Salt Solution
This solution was prepared by dissolving 0.028gm of (NH4)2Fe(SO4)2.6H2O,
0.2 gm of MgSO4.7H2O, 8 gm of NH4CL, 18 gm of KH2PO4, 30 gm of K2HP04
,in 750 ml of D.W then completed to 1000 ml. The pH of the solution was
Chapter two
Materials and Methods 43
adjusted to 7 and sterilized by autoclaving121° C for 15minutes (Vijve et
al.,1975)
2.1.9.7 Sodium thiosulphate ( 5 % )
This solution was prepared by dissolving 5 gm of Sodium thiosulphate in 90
ml of D.W then completed to 100 ml and sterilized by filtration through a 0.22
mm millipore membrane filter (Bautz and Freese, 1960).
2.1.9.8 McFarland solution (Tube No. 0.5)
Standard McFarland solution No.0.5 was prepared according to Baron et al.,
(1994 b) as follows:
Solution (A) was prepared by dissolving1.175 gm of barium chloride
(BaCl2.2H2O) in 90 ml of D. W then completed to 100 ml.
Solution (B) was prepared by adding 1 ml of concentrated sulfuric acid (H2SO4)
in 190 ml of D.W. then completed to 100 ml.
The two solutions were mixed by the adding 0.5 ml from solution A to 99.5
ml from solution B. The prepared solution was used to compare the turbidity of
bacterial suspension in order to obtain an approximate cell density of 1.5 ×108
CFU / ml.
2.1.9.9 Phosphate Buffer Saline (PBS)
This buffer was prepared according to Poxton and Brown, (1996) by dissolving
12.1 gm of K2HPO4 (anhydrous), 3.4 gm of KH2PO4 (anhydrous), 0.8 gm of
NaCl in 950 ml of D.W. The contents were Mixed to dissolve, the pH was
adjusted to 7.2. Then the volume was completed to 1000 ml of D.W and
autoclaved at 1210C for 15 minutes, and then stored this solution at 40C.
Chapter two
Materials and Methods 44
2.1.9.10 Sorensen’s Phosphate buffer (Leishman stain buffer)
This buffer was prepared according to Poxton and Brown, (1996) ; Jaypee
,(2000) as follow:
Solution I: prepared by dissolving 9.1 gm of KH2PO4 in 1000 ml of D.W.
Solution II: prepared by dissolving 9.5 gm of Na2HPO4 in 1000ml of D.W.
A volume of 43.5 ml of Solution I was taken and completed to 100 ml by using
Solution II, the pH was adjusted to 6.8.
2.1.9.11 Solution for mobile phase of thin layer chromotograph
The mobile phase of TLC was prepared according to Nakashima et al., (2005)
,which contains benzene- methanol- acetic acid 87:11:2 (vol/vol/vol).
2.1.9.12 Seeds extract
It was prepared by dissolving 5 gm of milled seeds ( peanut , sunflower,
sesame ) in 90 ml of D. W. them the volume completed to 100 ml
2.1.10 Solutions for DNA extraction
2.1.10.1 Tri-Ethylene Diamine Tetra acetic Acid (TE buffer)
This solution was prepared by dissolving 0.2420 gm of tris-base and 0.0744
gm of Na2-EDTA in 90 ml D.W , pH was adjusted to 8.0 , then the volume
completed to 100 ml ,autoclaved at 1210C and stored at 40C (Pospiech and
Neuman , 1995).
2.1.10.2 Tris- Boric acid- Ethylene Diamine Tetra Acetic Acid (TBE) buffer
This solution was prepared by dissolving 3.02 gm of Tris – base, 0.19 gm of
Na2-EDTA and 1.5 gm of boric acid in 450 ml of D.W , pH was adjusted to 8.0.
Chapter two
Materials and Methods 45
Then the solution was completed to 500 ml of D.W, sterilized by autoclaved at
1210C for 15 minutes and stored in 40C ( Lema et al .,1994).
2.1.10.3 Sodium Dodesyl Sulfate solution (SDS 10%)
This solution was prepared by dissolving 1 gm of SDS in 10 ml of D.W.
2.1.10.4 Lysozyme solution (30mg/ml)
This solution was prepared by dissolving 0.030 gm of lysozyme in 1ml of
D.W.
2.1.10.5 Ethidium bromide(10mg/ml)
This solution dye was prepared by dissolving 0.5 gm of ethidium bromide
powder in 1 ml of absolute alcohol , the volume was completed with distilled
water to
10 ml and placed in dark container and stored in 40C for use
( Sambrook et al., 1989).
2.1.10.6 Sodium chloride solution (5M)
This solution was prepared by dissolving 7.3 gm of sodium chloride in 25 ml
of D.W , autoclaved at 1210C for 10 minutes , and stored at 40C.
2.1.10.7 Saline EDTA - Tris Hcl (SET) buffer
This solution was prepared by dissolving 0.4 gm of sodium chloride , 1.4 gm
of Na2EDTA and 0.24 gm of tris-base in 90 ml of D.W . The pH was adjusted to
8.0 , and the solution was completed to 100 ml of D.W .Then autoclaved at
1210C for 15 minutes and stored at 40C (Pospiech and Neuman, 1995).
Chapter two
Materials and Methods 46
2.1.10.8 Loading buffer
This solution was prepared by dissolving 0.025 gm of bromophenol blue ,
0.25gm of xyline and 1.5 gm of ficol in 10 ml of D.W . Mixed well and placed in
a dark container, and then stored at 40C (Sambrook et al.,1989).
2.1.10.9 Agarose gel (0.8%)
It was prepared by dissolving 0,8 gm of agarose in 90 ml TBE buffer ( section
2.1.10.1) and completed to 100 ml ,melted by boiling in microwave and then
cooled to 550 C.
2.1.11 Laboratory animals
Twenty healthy Balb /C male mice were obtained from National Center For
Drug Control and Research (NCDCR) .The animals were kept in the animal
house of the university of Al- Nahrane . Mice were (6-8) weeks old, weighing
approximately 20 gm. They were placed in cages measuring (29×15×12) cm.
Food, Water, aeration and cleaning of the cages were a achieved of daily. All
these animals were used in the experiment of animal infection by S. aureus
2.2 Methods
2.2.1 Collection of specimens
In this study, 207 clinical samples were collected from out and in patients,
(males and females) with different ages, who suffered from different diseases
such as wounds, chronic suppurative otitis media, urinary tract infections (UTIs).
vagainal infection(from females), eye infection, nasal infection and bacterimia.
Chapter two
Materials and Methods 47
The patients were attended
from centric kids technical hospital, AL-Shafa'
Hospital during the period of December / 2011 to May / 2012.
2.2.2 Characterization of specimens
The samples were collected according to the methods suggested by Laitinen
et al .,(1994) ; Alnoso et al., (1999) ;MacFaddin ,(2000) and Zhanle et al.,
(2000). In case of ear, eye, nose, vaginal infections, swabs moistened with BrainHeart Infusion broth (BHI) (section 2.1.5.1) or saline. In case of wounds, swabs
were taken from the deep lesion with a dry swab moistened with a little amount
of (BHI) broth or saline after cleaning with 70% ethyl alcohol.
In case of UTI mid stream urine were generally collected in plastic universal
sterile containers. In case of blood sampls, blood was collected from patients by
sterile syringe and placed in glass universal sterile tubes containing (BHIB).All
swabs and specimens were transported to the laboratory without delay. The
samples were immediately inoculated in Mannitol Salt agar , Nutrient agar
( section 2.1.5.1) and blood agar (section 2.1.5.2) and incubated for overnight at
37 0C under aerobic conditions (Table 2-9).
Table (2-9) Clinical samples collected from patients with different infections
Clinical
Specimen
Collecting tool
Urine
Containers
Ear infection
Ear swabs
Swabs
Eye infection
Eye swabs
Swabs
Blood
Tubes
Wound infection
Wound swabs
Swabs
Vaginal infection
Vaginal swabs
Swabs
Nasal swabs
Swabs
Urinary tract infections
Bacterimia
Nasal infection
Chapter two
Materials and Methods 48
2.2.3 Bacterial isolation
The colonies appeared on nutrient agar , blood agar and mannitol salt agar
were selected for further diagnostic tests. Diagnosis of S. aureus. was determined
according to Bergey’s manual of systematic bacteriology (William et al., 2009).
2.2.3.1 Cultural characteristics
These characteristics include; colonial morphology ,size of colony, color and
the affect on the media such as blood hemolysis, pigments appear on Milk agar
and ability to ferment mannitol ( William et al., 2009).
2.2.3.2 Microscopic Examination
This include the examination of shape, Gram-stain reaction and arrangement of
cells .
2.2.3.3 Biochemical Tests
2.2.3.3.1 Oxidase Test
A filter paper was moistened with several drops of freshly prepared oxidase
reagent (1%), and then a small portion of the colony to be tested was picked up
and placed on the moistened filter paper. No change in color within 30 seconds
indicates a negative test (Collee et al., 1996).
2.2.3.3.2 Catalase Test
Few drops of 3% H2O2 were dropped onto a small portion of the culture to be
tested, placed on a clean dry slide, the formation of bubbles indicates a positive
test (Collee et al., 1996).
Chapter two
Materials and Methods 49
2.2.3.3.3 Coagulase test
This test was used to detect bound coagulase and free coagulase,
(Baron et al., 1994a).
2.2.3.3.3* Slide Coagulase test (Slide method)
Slide test was used to detect the bound coagulase enzyme by mixing one drop
of sterile distilled water with one colony of bacteria on a glass slide then one
drop of human plasma was added and mixed carefully and the result was read
after 10-15 minutes. A positive and negative controls were prepared by the same
way for comparison.
2.2.3.3.3** Coagulase test (Free tube method)
In this test one colony of bacteria was inoculated with one milliliter of human
plasma ( diluted by normal saline 1:6 ) in a test tube which then incubated at
37oC for 24 hour . The tubes were examined after 1-4 hours .
The positive result is a clot formation but when result is negative the tube was
reincubated for 24 hour and examined again.
2.2.3.3.4 Blood Hemolysis Test
Blood agar medium (section 2.1.5.2.1) was streaked with the bacterial culture
to be tested and incubated at 370 C for 24 hours. The appearance of a clear zone
around the colony indicates ß–hemolysis while the presence of green-color
indicates α–hemolysis (Baron et al., 1994b).
Chapter two
Materials and Methods 50
2.2.3.3.5 Urease test
A loopful of bacterial culture was inoculated on slant agar-containing urea
(section 2.1.5.1) and incubated at 370 C for 24hr. Urease positive cultures are
changed the color of the indicator from yellow to purple-pink (Collee et al.,
1996).
2.2.3.3.6 Degredation of nucleic acid
A loopful of bacterial culture was inoculated on DNase medium (section
2.1.5.2.3) and incubated at 370 C for 24 hr .The positive result appeares as a clear
zone around the colony.
2.2.3.3.7 Protease production test
A loopful of bacterial culture was inoculated on milk agar (section 2.1.5.2.4)
and incubated at 370 C for 24 hr . The positive result appeares as a clear zone
around the colony.
2.2.3.3.8 Gelatinase hydrolysis test
A loopful of bacterial culture was inoculated on gelatin agar (section 2.1.5.2.2)
and incubated at 37Co for 24 hr , The positive result turns gelatin to liquid in
refrigerator.
2.2.3.3.9 Sugar fermentation Test
Plates containing medium prepared for carbohydrates fermentation
(section 2.1.5.2.11) were inoculated with 0.1 ml of bacterial culture and
incubated at 370 C for 24 hr . Production of yellow color indicates a positive
result.
Chapter two
Materials and Methods 51
2.2.3.4 The characterization of the isolates using Api staph.
Api- 20 Staph is a standardized system combining 20 biochemical tests that
offer widespread capabilities. It enables group or species identification of most
Staphylococcaceae species .
2.2.3.4.1 Preparation of the inoculums.
A single pure colony cultured aerobically on blood agar medium for 18 -24hr
was transferred to the test tube which contained 5 ml of normal saline ( section
2.1.9.4). Mixed well by using the mixer . The turbidity of bacterial suspension
was compared with McFarland standard no. 0.5 ( section 2.1.9.8) .
2.2.3.4.2 Preparation of the strip
* An inoculum box (Tray and Lid) was prepared by distributing 5ml of sterile
distilled water to the wells of the tray to provide the humidity for bacterial growth.
* The strip was removed from it's package and placed in the tray.
* The strip was then inoculated with bacteria by a sterile Pasteur pipette.
* Anaerobiosis was made in the ADH and URE test by filling the wells with
mineral oil to form a convex meniscus.
* The tray was then closed and incubated at 35oC – 37oC for 18-24 hour.
2.2.3.4.3 Addition of reagents
After the incubation period was finished, the following reagents were added :
VP test : One drop of reagent VP1 (40 % potassium hydroxide ), then one drop
of reagent VP2 (5% alfa- nephthol ).
NIT test : One drop from NIT1 and NIT2 .
PAL test : One drop from ZYMA and ZYMB reagents .
Chapter two
Materials and Methods 52
2.2.3.4.4 Reading the results
- After 10 minutes from the addition of the reagents, the results were read
according to the (Appendix-1) supplied by the Api Staph system .
- The results were recorded on the results sheets .
- after words the identity of test bacteria ( Genus and Species) was interpreted
using the analytical profile index supplied by the manufacturing company (BioMerieux).
2.2.4 Maintenance of bacterial isolates
The maintenance of bacterial isolates were performed according to Collee and
Marr (1996) as follows:
2.2.4.1 Short-term storage
Bacterial colonies were maintained for a period of few weeks on the surface of
Mannitol Salt agar medium (section 2.1.5.1). The plates were tightly wrapped
with parafilm and stored at 40 C.
2.2.4.2 Medium-term storage
Bacterial isolates were maintained by streaking on Brain-Heart agar slant
(section 2.1.5.1) for a period of few months. Such medium was prepared in
screw-capped vials containing 10-15 ml of the medium. The isolates were
streaked on the slant and incubated at 370 C for 24hours. The slants with well
grown colonies were wrapped with parafilm and stored at 40 C.
2.2.4.3 Long-term storage
For preservation of a bacterial isolate for a long time, a sterile tube containing
2ml of nutrient broth (section 2.1.5.1) containing 15% glycerol was inoculated
Chapter two
Materials and Methods 53
with the isolate and incubated at 370 C for 24 hours. When visible bacterial
growth was seen (turbidity), the tube was sealed tightly with parafilm and stored
at -200 C.
2.2.5 Viable count determination of the bacterial inoculum
The relationship between viable count and the optical density was determined
according to Poxton and Brown , (1996) as follows :
· The selected S. aureus isolate was grown on Brain – Heart infusion agar
medium (section 2.1.5.1).
· A loopful of several pure single colonies were collected by using a sterile
loop and placed in a test tube containing 10ml normal saline ( section
2.1.9.4), and mixed by a mixer .
· Different five turbidity tubes were prepared from the bacterial suspension
,then the optical density was measured for each tube at wave length of 620
nm.
· The viable count was measured by performing decimal dilution for each
tube , then 0.1 ml of each diluent was spread on plate count agar medium (
section 2.1.5.1) using L-shaped spreader, duplicate was done for each
diluents , then the plates were incubated at 37oC for 24 hr.
· The vaiable count was measured according to the following equation :
Viable count / ml = number of colonies × dilution factor ×10
· Finaly the relation between the viable count and the optical density (O.D.)
was established and a standard curve was drawn.
Chapter two
Materials and Methods 54
2.2.6 Susceptibility test for antimicrobials
The susceptibility of S. aureus isolates to different antimicrobials were
determined according to Kirby-Bauer disk diffusion method (Bauer et al., 1966)
as follows:
2.2.6.1 Preparation of culture media
Mueller-Hinton medium (section 2.1.5.1) was employed for this experiment.
The medium was autoclaved at 1210 C for 15 minute, cooled to 45-50°C and
distributed in sterile Petri dishes.
When the medium solidified, the Petri-dishes were incubated at 370 C for 30
minutes to evaporate the excess moisture.
2.2.6.2 Preparation of the inoculum
With a sterile wire loop, the tops of 4-5 pure colonies were transferred to a tube
containing 5 ml of BHI broth ( section 2.1.5.1) and incubated at 370 C until the
appearance of turbidity. This usually required at least 4–6 hours of incubation (
OD620 was measured and fixed on 0.5).
2.2.6.3 Inoculation
A volume of 100 µl from the bacterial isolate was placed on MHA(section
2.1.5.1). A sterile swab was then used to streak the dried surface of a Mueller–
Hinton plate in 3 different planes by rotating the plate approximately 60 degrees
each time to obtain a distribution of the inoculum.
2.2.6.4 Application of the disks
With a sterile forceps, the selected antimicrobial disks were placed on the
surface of the medium with a constant distance from each other and pressed
Chapter two
Materials and Methods 55
firmly but gently into the agar with sterile forceps. The inoculated plates were
incubated at 370 C for 18 hours in an inverted position.
2.2.6.5 Reading the Results
After incubation, the diameters of the inhibition zones were noted and
measured by using a ruler. The results of this experiment were recorded
according to the standard guidelines recommended by the National Committee
for Clinical Laboratory Standard. The measured diameters were compared with
zones of inhibition (Bauer et al.,1966; CLSI,2006; NCCLs, 2007)(Appendix-2).
2.2.7 Detecting the ability of S.aureus isolates to produce
staphyloxanthin
In order to identify the ability of S.aureus isolates for staphyloxanthin
production , 10 ml of BHIB was inoculated with 100μl of S.aureus isolate and
incubated at 37°C for 18 hour in order to get 10 8 CFU/ ml (Bacterial growth
was determined by measuring the absorbency at 620 nm). A volume of 100 μl of
the inoculum from each isolate
was streaked on
different culture media
including: Milk agar medium ,Peanut seeds medium , Sunflower seeds medium,
Sesame seed medium , Tripticase yeast medium, Carotinoid expression medium
and Tripticase soya medium incubated at 37°C for two days and then incubated
at 20°C for two days . Appearance of growth with pigment (orange ,yellow )
indicates a positive result (Grinsted and Laccy,1973). According to the pigment
production , the isolates were selected.
Chapter two
Materials and Methods 56
2.2.8 Extraction of pigment staphyloxanthin (STX)
Four different extraction methods were used as explained in the following:
2.2.8.1 Extraction by acetone
The pigment of Staphylococcus aureus (STX) was extracted according to the
method described by Giri et al.,(2004) as follows:
· Two isolates AE32 and AE36 were activated by growing on BHIB (section
2.1.5.1) , then the bacterial suspensions were adjusted to (0.5) measured at
OD620. A volume of 100 µl of this suspension was placed on milk agar
(section 2.1.5.2.4) with two repeats for each isolate. The plates were
incubated at 370C for 48 hr and then at 200C for 48 hr ,
· Agar surfaces were rinsed with sterile double distilled water (each rinse
with 3 ml).
· Bacterial cell were collected in sterile plan tube and added 5 ml of
sterilized distal water .
· The bacterial cells were centrifuged at 6000rpm for 15 min.
· The supernatant was discarded and the pellet was re suspended with
double distilled water and then centrifuged at 6000rpm for 15 min.
· The pellet was mixed of with 8 ml of acetone and wrapped with aluminum
foil to prevent exposure to light.
· Centrifuged at 10000rpm for 15 min.
· The packed cells were resuspended in 3 ml of acetone, held at 55°C in
water bath for 5 min and cooled for 10 min , and then the extract was
obtained by centrifugation at 10000rpm for 15 min..
Chapter two
Materials and Methods 57
· The extraction was repeated twice, until no further pigment could be
extracted.
· Carotenoids were estimated quantitatively by measuring absorbance of
solutions at 460 nm (maximum absorbance of the primary carotenoid
pigment staphyloxanthin extracted by acetone).
2.2.8.2 Extraction by chloroform
The pigment of Staphylococcus aureus (STX) was extracted according to
the method described by Ra’oof and Latif (2010) as follows:
· Culture sample (5ml ) was grown on milk agar plate (section 2.1.5.2.4).
· The growing cells were deposited using centrifugation at 10000 rpm for
15 min.
· The culture supernatant was transferred to a new test tube and 3 ml of
chloroform was added and mixed together
· The top layer was removed to a new test tube, and 1 ml of HCl (0.2 N)
was added.
· The test tube was centrifuged at 10000 rpm for 15 min, and the top layer
was transferred to cuvette tube. The absorbancen of this solution was
measured at 520 nm using spectrophotometer.
· A quantity of 1 ml NaOH (0.4 N) was added.
· The amount of Staphyloxanthin was transferred to a Petri dish, and was
left to a limited period (48 hr) to evaporate the chloroform in oven at
500C.
· The Staphyloxanthin powder was dissolved using sterile distilled water
Chapter two
Materials and Methods 58
2.2.8.3 Extraction by ethyl acetate
Extraction of staphyloxanthin pigment was done used on the
method of
Anurada et al.,(2004) as followed:
· Bacterial suspension was centrifuged at 10000 rpm for 15 min and the
supernatant was extracted with ethyl acetate (10 ml).
· The pigment from the pellet was extracted with acetone (10ml) and was
centrifuged at 10000 rpm for 15 minutes.
· The white pellet was discarded.
· Ethyl acetate fraction and acetone fraction were mixed and dried using
dialysis bag placed in a peaker containing sucrose.
· The extract was evaporated at room temperature.
· The residue was collected and stored at 40C until further use.
2.2.8.4 Extraction by methanol
The pigment of Staphylococcus aureus (STX) was extracted according to
method described by Marshall and Wilmoth (1981a),as follows:
· Bacterial cells were recovered from the growth on milk agar plate for 48hr
at 37oc then 48hr at 20oC.
· Agar surfaces were rinsed with sterile double distilled water (each rinse
with 3 ml).
· Bacterial cell were collected in sterile plan tube and added 5 ml of
sterilized distal water.
· Then the bacterial cells were centrifuged at 10000rpm for 15 min
· The supernatant was discarded and the pellet was resuspended with double
distilled water and then centrifuged again at 10000rpm for 15 min.
Chapter two
Materials and Methods 59
· Staphyloxanthin extraction from the pellet containing the bacterial cells
was carried out according to Dufosse et al.,(2001).
· The pellet was mixed with 8 ml of 99.9% methanol wrapped with
aluminum foil to prevent exposure to light.
· Centrifuged at 6000rpm for 15 min.
· The packed cells were resuspended in 3 ml of methanol, held at 55°C in
water bath for 5 min and cooled for 10 min, and then, the supernatant was
collected by centrifugation (10000 rpm for 15 min) again.
· The extraction was repeated twice, until no further pigment could be
extracted.
· Carotenoids were estimated quantitatively by measuring the absorbance of
the solution at 450 nm ( Lipovsky et al., 2008; Rosado et al.,2010;).
· The staphyloxanthin extract was evaporated by oven 550C for even
dryness.
2.2.9 Staphyloxanthin pigment assay
According to method described by Tao et al .,(2010),the staphyloxanthin
pigment was measured as follows :
After extraction of staphyloxanthin from two isolates (AE36 and AE32 ) by using
four different extraction methods (section 2.2.8), the absorbence of
staphyloxanthin was determined at 450 nm and the amount of the pigment was
calculated by using the following equation:
V (A - 0.0051)
Total carotenoids unit/cell =
0.175W
Chapter two
Materials and Methods 60
Where:A : Is the absorbance value of the diluted extraction at 450nm.
V: Is the final volume of the extract.
W(g): Is the weight of the dried powder.
0.175: Is the extraction coefficient of carotenoids
unit/cell : cuvate cell (for spectrophotometer).
2.2.10 Determination of optimal conditions for staphyloxanthin
production
The effects of some factors on pigment production were studied to determine
the optimum conditions for the production.
2.2.10.1 Effect of medium composition
A volume of 100µl of bacterial inoculum 105 cell/ml (section 2.2.5) was
cultured on different prepared media (section 2.1.5.2.4 → 2.1.5.2.10) and in
ready made media MHA, BHIA and N.A (section 2.1.5.1) .Two replicates were
made from each medium. The plates were incubated at 370C for 24 hr .The
pigment was determined after extraction by different methods. The concentration
of the pigment was determined by using the equation (section 2.2.9) after
extracted it.
2.2.10.2 Determination of optimum temperature for staphyloxanthin
production
After choosing the best media for pigment production, the optimum
temperature was determined by inoculating 100 µl of the bacterial inoculum 105
cell/ml (section2.2.5) on milk agar (section 2.1.5.2.4 ) .The plates were
incubated at different temperatures (20, 25, 28, 37, 40)0C for 24hr . The pigment
Chapter two
Materials and Methods 61
concentration was determined after extraction by methanol (section 2.2.8.4)
using the equation (section 2.2.9) after extracted it.
2.2.10.3 Determination of optimum pH for pigment production
Milk agar (section 2.1.5.2.4) was prepared in different pH values (5, 6, 7, 8, 9)
by using NaoH and HCL. A volume of 100 µl of bacterial inoculum 105 cell/ml
(section 2.2.5) was placed in the optimum medium and incubated at 370C for 24
hr , the pigment concentration was determined using the equation (section 2.2.9)
after extracted it.
2.2.10.4 Determination of optimum incubation time for pigment production
Milk agar (pH 8) (section 2.1.5.2.4 ) were inoculated by 100 µl of bacterial
inoculum 105 cell/ml (section 2.2.5) and incubated at 370C for different times
(18, 24, 36, 48, 72) hr .The pigment concentration was determined using the
equation (section 2.2.9) after extracted it.
2.2.10.5 Effect of aeration in staphyloxanthin production
Milk broth (section 2.1.5.2.12) was prepared in 250 flasks ml in different
volumes (50, 100 150) ml , at pH 8 . The prepared flasks were inoculated with
100 µl of bacterial inoculum 105 cell/ml (section 2.2.5) and incubated in a shaker
(160 rpm/min) at 370C for 72 hr . The pigment concentration was determined
using the equation (section 2.2.9) after extract it.
Chapter two
Materials and Methods 62
2.2.11 Partial purification of staphyloxanthin by thin layer
chromatography
Thin-layer chromatography (TLC) was used as an analytical and partial
purification . Staphyloxanthin was characterized by thin layer chromatography
using silica-gel (20cm x 20cm, Merck). The TLC plates was spotted with 4 drops
of staphyloxanthin extract ( final concentration 2 mg\ml) on the prepared line
near one end of the aluminum oxide plate , then plate was put in the jar contain
solvents: benzene-methanol-acetic acid 87:11:2 (vol/vol/vol)(section 2.1.9.11)
which act as a mobile phase. The solvents were moved until they reached near
the upper of plate then it was removed from the jar and allowed to dry. Most
carotenoids are readily detected as colored spots or bands. Other compounds of
pigment were colorless but fluorescents strongly in UV light these colorless
components can also be detected by placing the plates in a closed jar saturated
with iodine vapors. The Rf value was calculated according to the following
equation
Rf =
Distance of spot sample movement
Distance of spot solvent movement
After developing, the spots from thin-layer plate were recovered by scraping
the appropriate portion of silica gel into a tube and eluting with
methanol.(Marshall and Willmoth,1981a ; Wieland et al.,1994) .
Chapter two
Materials and Methods 63
2.2.12 Staphyloxanthin as antibacterial agent
The activity of staphyloxanthin as antibacterial agent was tested by using the
well-diffusion method according to the method described by Samaranika ,2012
as follows:
· The MHA plates (section 2.1.5.1) were
(section
2.2.5)
Pseudomonas
of
indicator
putidae,
isolates
Pseudomonas
inuculated with 108cell/ml
(Pseudomonas
fluorescence,
aeruginosa,
Staphylococcus
epidermidis, Escherichia coli, Klebsiella spp, Salmonella spp, Shigella
spp., Proteus spp.)
· Wells were prepared in the plates with 6 mm sterile cork borer.
· The wells for each culture of indicator bacteria were filled with 100 µl of
staphyloxanthin solutions ( final concentration 0.2 g / ml) extracted from
isolate AE36 and AE32 .
· The plates were incubated at370C for 24 hr.
· Inhibition was detected by a zone of clearing around the partial purified
staphyloxanthin extract.
2.2.13 The induction of mutants with chemical agents
2.2.13.1 Mutation by ethyl methane sulfonate (EMS)
· Chemical mutagenesis of locally isolated S.aureus AE36 was performed by
incubation of this isolate with the chemical mutagen ethyl methane
sulfonate( EMS ) according to the procedure described by
Bautz and
Freese, (1960) and Vijver et al,.(1975) as follows :
· A bacterial inoculum 1×107 CFU/ml was added to 10 ml of TSB
,incubated at 37oC for 72hr.
Chapter two
Materials and Methods 64
· The EMS was diluted by stock salt solution (section 2.1.9.6)(33µl :10ml)
(vol/vol).
· A quantity of 20µl of the diluted EMS solution was added to 1.980 ml of
culture media (the final concentration was 10µg/ml).
· A quantity of 50 µl of bacterial inoculum was added .
· The culture was incubated at 37°C for 24hr and then centrifuged at 4000
rpm/min,
· The alkylation process was blocked by washing the pellet with 2ml of 5 %
sodium thiosulphate ( section 2.1.9.7)
· The suspension was centrifuged at 4000 rpm the supernatant
was
discarded. The pellet was resuspended with 5ml of TSB ,incubated at 37oC
for 72 hr.
· A quantity of 100 µl was taken and grown on milk agar for 48hr at 37oC
and then for 48hr at 20oC to visualized the mutant cell .
2.2.14 The use of Staphyloxanthin pigment as anti oxidant with
whole blood cell and neutrophils invitro
The anti oxidant affect of staphyloxanthin pigment was detected using whole
blood cell and neutrophils according to the method described by Liu et al.,(2005)
as follows :
· The bacterial isolate was activated on TSB for 72hr at 37oC .
· The suspension was centrifuged at 8000 rpm for 10 min and the
supernatant was discarded . The pellet was washed twice in 2 ml of PBS
( section 2.1.9.9 ) then centrifuged at 8000 rpm for 10 min .
· A volume of 5 ml PBS was added to the pellet.
Chapter two
Materials and Methods 65
· The inoculum was diluted to 105 CFU by using PBS (2:6) (Vol / Vol).
· The inoculum was mixed with 6 ml of freshly drawn human blood (1 : 6)
(vol:vol) in heparinized tube and incubated at 37oC for 4hr .
· One drop of blood was spread on a slide and left to dry on a filter paper at
room temperature .
· The slide was covered by leishman’s stain for 3 min and then covered by
phosphate buffer sorensen (section 2.1.9.10) and mixed well for 7 min to
mix stain with the buffer.
· The slide was rinsed under tap water for 3min and dried by filter paper.
· The slide was Examined under oil emersion (X100) after the addition of
one drop of oil on the slide and the neutrophils were counted.
2.2.15 Study the effect of S. aureus (pigmented, non pigmented and
mutant ) isolates and treatment in an animal model
2.2.15.1 Preparation the bacterial suspension
In this experiment the bacterial suspension of S. aureus was prepared by
growing the bacterial isolate in TSB ( section 2.1.5.1) , incubated overnight at
37oC. Then the turbidity was standardized to the concentration of 1×107 CFU/ml
(Bunce et al.,1992;Nizet et al.,2001).
2.2.15.2 Laboratory animals used
In this experiment 20 mice were used ,they were (6-8) weeks old and
weighting 20 gm as mentioned in section (2.1.11).
Chapter two
Materials and Methods 66
2.2.15.3 Experimentally infected mice with bacteria and treatment with
Ciprofloxacin
In this experiment, the pathological effect of the bacterial suspension was
studied, The animals were infected by skin lesion and the results were compared
with control mice .Four groups of mice were used , every group includes 5
animals as follows:
Group1 (group control): in this group 5 animals received saline (section
2.1.9.4), by askin lesion.
Group 2: Animals were received 1×107 CFU/ml of yellow pigmented S. aureus
AE36 producer from day 0 to day 4 and then treated with Ciprofloxacin ointment
from day 5 to day 8.
Group 3: Animals were received 1×107 CFU/ml of white pigment S. aureus
AE38, from day 0 to day 4 and then treated with Ciprofloxacin ointment from
day 5 to day 8.
Group 4: Animals were received 1×107 CFU/ ml S. aureus mutant ∆AE36 of
pigmentation from day 0 to day 5 and then treated with Ciprofloxacin ointment
after the appearance of infection until complete healing.
Wound swab was taken from the animals of the groups (2,3,4) and was spread
on plate count agar medium (section 2.1.5.1)using L-shaped spreader. Duplicate
was done for each diluents , then the plates were incubated at 37oC for 24 hr.
Chapter two
Materials and Methods 67
2.2.16 Extraction of bacterial DNA
2.2.16.1 Extraction by salting out
The DNA was extracted from the bacterial isolates (AE38, AE36, ∆AE36) by
salting out method according to method described by Pospiech and
Neuman,(1995) as follows :
· A quantity of 5 ml sample of culture grown in TSB incubated at 37oC for
24 hr was centrifuged using cooling centrifuge at 10000rpm for 15 min at
4oC.
· A volume of 500 µl of SET (section 2.1.10.7)was added to the pellet and
mixed well by mixer. Then centrifuged again at 10000 rpm for 15 min at
4oC ,then resuspedned with 1.5 ml of SET buffer .
· A volume of 100 µl lysozyme (section 2.1.10.4 ) was added to SET buffer
and mixed well and then incubated at 37oC for 1 hr.
· A volume of 200 µl of SDS was added (section 2.1.10.3 ) mixed by hand
for 5 min , then incubated at 56oC for 1 hr in a water bath .
· The tubes were cooled at room temperature , then 2 ml of sodium chloride
(section 2.1.10.6) was added and placed in a room temperature for 10 min.
· A volume of 500µl chloroform was added and mixed for 30 min , then
centrifuged using cooling centrifuge at 12000 rpm for 20min at 4oC.
· The aqueous phase was transferred to clean new tube and a double volume
of cold ethanol was added.
· The tube was placed in a freezer at -20oC for 18 hr.
· The tube was centrifuged at 10000rpm for 20min at 4oC . The supernatant
was discarded and the precipitate was left to dry completely then dissolved
in 100µl of TE buffer (section 2.1.10.1).
Chapter two
Materials and Methods 68
2.2.17 Estimation of the DNA concentration
· Five microliters (µl) of each sample AE36 , AE38, ∆ AE36 were added to
495µl of TE (Tris-EDTA) then mixed well to determine the DNA
concentration and its purity. As blank TE buffer was used.
Aspectrophotometer was used to measure the absorbance at wave length
of 260nm and 280nm. An O.D of 1 corresponds to approximately 50
µg/ml for double stranded DNA .
· The concentration of DNA was calculated according to the formula:DNA concentration (µg/ml) = O.D 260nm´50´Dilution factor
· The spectrophotometer was used also to estimate the DNA purity ratio
according to this formula:DNA purity ratio = OD260/OD280
· This ratio was used to detect nucleic acid contamination in protein
preparations. DNA quality was also assessed by analyzing the DNA using
agarose gel electrophoresis (Sambrook et al., 1989).
2.2.18 Agarose gel electrophoresis (Sambrook et al., 1989)
DNA was detected by using agarose gel electrophoresis as follows :
· Agarose at a concentration of 0.8 % was prepared by dissolving 0.8 gm of
agarose in 100 ml of TBE buffer (section 2.1.10.1), and melted by boiling
in a microwave, then cooled to 55oC.
· A volume of 5 µl Ethidium bromide dye (section 2.1.10.5) was added to
melted agarose ( final concentration 0.5 µg / ml).
Chapter two
Materials and Methods 69
· The tray was prepared and the comb was placed to make well adjacent at
the end of tray and leave a suitable distance, infusion of the gel in the tray
was gently carried out.
· The comb was gently lifted after assuring the rigidity of the gel.
· The tray was transfered to the gel electrophoresis unit which was filled
with TBE buffer in a way to overflow gel surface by 1 ml .
· The sample of DNA was prepared by mixing 10 µl of DNA with 5 µl of
loading buffer (section 2.1.10.8). Then loaded in the well.
· The stained agarose was poured into a tray sealed previously with a tape,
the lid of the tank was closed and the electrical leads were attached to a
power supply, hence the DNA bands migrate towards the positive pole
(anode), and the 5 Volt/cm electrical voltage was applied.
· The electric current was passed through the gel at 45 Volt for the first 15
minutes and at 70 Volt for 1.5 – 2 hours.
· DNA band was examined by using gel documentation apparatus to
analyze the results.
2.2.19 Real-Time PCR technique
Real-time PCR is a quantitative PCR method which used in this study to
detect staphyloxanthin pigment and selected Methicillin-Resistant gene in the
Staphylococcus aureus (MRSA) isolate via using SYBR Green RT-PCR as
follows
The extracted DNA , primers and PCR Master Mix ( Bioneer \ Korea ), were
thawed at 4˚C ,mixered and centrifuged briefly to bring the contents to the
bottom of the tubes . For each run a duplicate of each sample was prepared in
Chapter two
Materials and Methods 70
addition to a negative control (test for the presence of contamination or the
generation of nonspecific amplification products under the assay conditions
used). In order to reduce the risk of false-positive tests. The negative control
contained the same material except that 3.0 µl of D.W was added instead of
template DNA .
2.2.19.1 SYBR Green real-time PCR assay
Staphyloxanthin pigment and Methicillin-Resistant Staphylococcus aureus
(MRSA) was determined according to methods described by Liu et al.,( 2005)
and Suhaili et al ., (2009) as follows :
* Primer Sequences
crtM-F
5¢- TTA GGA AGT GCA TAT ACT TCA C -3¢
crtM-R
5¢- GGC AAC GTT ATA CGA TCA TCG T -3¢
mecA-F 5¢- GAT TAT GGC TCA GGT ACT GCT ATC C -3¢
mecA-R 5¢- ATG AAG GTG TGC TTA CAA GTG CTA A -3¢
2.2.19.2 The procedure
· A volume of 10x primer was prepared that contain target-specific primer
for methicillin resistant Staphylococus aureus and pigment production
duplex PCR consists of 1pmol/μl(crtM primers) and 10pmol/μl (mecA
primers) in TE buffer.
· The reaction mixture was prepared according to table (2-10 ).
· The reaction mixture was mixed thoroughly, and 47 ml was dispensed into
PCR tubes .
Chapter two
Materials and Methods 71
· An aliquot of 3 ml DNA template was added to the every PCR tubes, then
the tubes were capped .
· The tubes were centrifuged briefly to spin down the contents and eliminate
air bubbles from solutions .
· The applied biosystems Real-Time 7500 PCR System was programmed
according to table( 2 -11).
· The PCR tubes were placed or plated in the real-time cycler, and start the
cycling program.
· Data analysis a melting curve was generate using applied biosystems Realtime PCR system software
2.2.20 Statistical Analysis
The optimization of staphyloxanthin production and neutrophils count, were
analyzed using the SPSS statistical software package and Microsoft Office Excel
, the results were analyzed by one way analysis of variance ( ANOVA ) . The P
values < 0.05 were considered significant . Data are presented as mean standard
deviation (S.D.) (SPSS. 2014).
Chapter two
Materials and Methods 72
Table ( 2- 10).Reaction Setup in Real Time PCR.
Volume (μl) per
Reaction Component
Final Concentration
Sample (3µl DNA)
SYBR®
Green
PCR
Master Mix (2X)
25
10X Primer Mix 1
1
10X Primer Mix 2
1
Nuclease-free Water
Total Volume
1X
10pmo/lμl crt M F primer
10pmol/μl crt M R primer
10pmol/μll mecA F primer
10pmol/μll mecA R primer
20
-
47
-
Table( 2-11) The program used in Applied Biosystems Real-Time 7500 PCR System.
Step
Enzyme Activation
Temperature
Time
Number of
(°C)
(min:sec)
Cycles
95
10:00
1
Denaturation
95
00:15
40
Annealing/Extension
65
01:00
Extension step
72
01:00
Amplification
Chapter three
Results and Discussion
73
Results and Discussion
3.1 Identification of Staphylococcus aureus
All clinical (207) bacterial isolates were characterized according to Bergey’s
Manual of Systematic Bacteriology (William et al., 2009) as well as, other
characters reported by Baron and Finegold,(1990); Baron et al., (1994a); Collee et
al.,(1996); Gorbach et al.,(1998); Macfaddin,(2000) and Brooks et al.,(2001).
Cultural , morphological and biochemical characteristics , revealed 43 isolates
(20.77%) being S. aureus. These isolates were marked by the letters AE. The
preliminary cultural diagnosis for bacterial isolates exhibited that all 43 isolates
were characterized by raised , smooth, glistening, translucent with varied
pigmentation production . the pigment was diagnosed on milk agar , trypticase soya
agar and trypticase yeast agar . All isolates were grown on (7%) human blood agar.
Some isolates showed β- hemolysis (95.4%) and some others showed α- hemolysis
(4.6%) . All isolates grew on mannitol salt agar and fermented mannitol . Under the
microscope ,the bacterial cells appeared as cocci arranged in clusters , positive for
gram stain reaction , non motile , non spore forming .
The results of the biochemical test (Table 3-1) were compared with the
characteristics of S. aureus documented by William et al., (2009) and others
( Baron and Finegold,1990; Baron, et al ., 1994a; Collee et al.,1996; Macfaddin,
2000). The bacteria produced water soluble pigments, hence it’s detection was
sufficient for identification of S. aureus . Moreover, all pigment producing S.
aureus are able to grow in 37oC, such capability was regarded as confirmative
character to S.aureus. All these observations are considered through the
identification of S. aureus isolates which were diagnosed in this study.
Chapter three
Results and Discussion
74
Table (3-1) The biochemical tests of Staphylococcus aureus
Test
Oxidase
Catalase
Hemolysis
Coagulase
Growth on NaCl agar:10%
Deoxyribonuclease (DNase test)
Protease
Urease
Gelatin liquefaction
Mannitol fermentation
Mannose fermentation
Melebioze fermentation
Raffinose fermentation
Sucrose fermentation
Trehalose fermentation
Xylose fermentation
Arabinose
Result
_
+
β and α
+
+
+
+
+W
+
+
+
_
_
+
+
_
_
(+) positive test , (-) negative test , + w (positive to weak reaction)
S. aureus cause broad spectrum of infection which is associated with nasal , eye,
blood, and ear infections ( Herfindal and Gourley , 2000).Table (3-2) shows the
presence of S. aureus according to the site of infection . The results indicated that
the percentage of S.aureus was 20.77% from the total samples (207) in this study
.Nasal infection (40%) was the most accessible site for S. aureus and this agrees
with the results of Abdalla et al .,(1998) who found that the percentage of S. aureus
in nasal infection was ( 39.1% ). This may be due to the high distribution of this
organism around the hospital environment (Michael et al., 2012). In addition , these
results are in agreement with the results obtained by other studies ( Cespedes et al.,
Chapter three
Results and Discussion
75
2002 (22.2%) ; Akoua et al.,2004(19.4%); Ogeer,2006 (20.1%)). Furthermore, the
present study revealed that the presence of S. aureus in eye samples(3.7%) were
less than in blood(37.14%), nose(40%), wounds (21.87%),
urine(19.35%),
vagina(15.15%) and ear infections(10.3%) .These results are compatible with the
results reported by Sefani and Varaldo,(2003) who mentioned that the presence of
S. aureus in eye samples were less than in other samples.
The differences in the percentage of infection according to the site obtained in the
present study may be due to the fact that these samples were not taken from the
same site. However, these results are still agree with those that says S. aureus is an
opportunistic pathogen that causes human infections and can be isolated from
different sites of infections ( Todar , 2008). Whereas the percentage of clinical
infection with S. aureus obtained in other studies were 21.87% (Suo’d ,2005),
30.1% (Albaldawi ,2005) , 18% (Rasheed ,2006) and 3.01% (Mahmood ,2006)
results.
Table (3 -2 ) Frequency of S. aureus according to the site of infection
Type of samples Total
Blood
Nose swab
Wounds swab
Urine
Vaginal swab
Ear swab
Eyes swab
Total
35
20
32
31
33
29
27
207
No. of
isolates
13
8
7
6
5
3
1
43
Percentage
%*
37.14
40
21.87
19.35
15.15
10.3
3.70
Percentage
%**
30.23
18.60
16.28
13.95
15.15
6.98
2.33
100%
Percentage
%***
6.28
3.86
3.38
2.89
2.42
1.45
0.48
*Percentage of S. aureus according to source ** Percentage of S. aureus according to total no. of
S. aureus isolate *** Percentage of S. aureus according to total no. of total sample.
Chapter three
Results and Discussion
76
3.2 Standard viable count curve of Staphylococcus aureus
To estimate the number of viable bacterial count /ml in bacterial culture, the OD
was measured and the viable count was estimated using five different turbidity
tubes of the isolates AE36 and AE38 grown for 18 hours. The relationship between
the viable count and the optical density was drawn as illustrated in Figure ( 3-1 )
Optical Density at 620 nm
and Figure ( 3-2 ) .
Figure (3-1) A standard curve of S.aureus AE36 isolate showing the relationship between the
optical density and the viable bacterial count/ ml.
Results and Discussion
77
Optical Density at 620 nm
Chapter three
Figure (3-2) A standard curve of S.aureus AE38 isolate showing the relationship between the
optical density and the viable bacterial count/ ml.
3.3 Api staph test
The biochemical identification of the bacterial isolates (AE36 and AE38) was
confirmed using the system of Api 20 for Staphylococcus , which contain 20
biochemical tests (Butler et al.,1975). The results were obtained after 24 hr of
incubation at 37oC . The results confirmed the earlier identification of the genus S.
aureus as shown in table (3-3), figure (3-3) .
Table (3-3) Results of Api20-Staph for S.aureus AE36 isolate
-
0
-
GLU
+
FRU
-
MNE
+ -
MAL
+
LAC
TRE
-
MAN
-
XLT
PAL
-
MEL
VP
+
NIT
RAF
-
XYL
+ +
SAC
MDG
NAG
ADH
URE
+
+
+
+
+
-
Chapter three
Results and Discussion
78
Figure (3-3 ) Api-20 Staph for identification of S. aureus AE36 Isolate
3 .4 Antimicrobial susceptibility of S. aureus
The antimicrobial susceptibility of S. aureus isolates ( Figure 3- 4 ) showed that
43 isolates were completely resistant (100%) to Amoxicillin/clavulanic acid,
Aztreonam, Carbenicillin, Ceftaxime, Cefotaxin, Cifixime, Nitrofluranton,
Pencillin G, Pipracillin and Vancomycin .Whereas Impenem and Trimethoprim
were the most effective drugs used in the present study. The AE36 isolate was
completely resistante to Amikacin, Amoxicillin/clavulanic acid ,Ampicillin,
Aztreonam, Carbenicillin, Ceftaxime, Cefotaxin, Ceftrazidime, Ceftriaxone,
Cifixime, Nitrofluranton, Pencillin G, Pipracillin and Vancomycin . While AE38
isolate showed resistante to Amikacin AK Ceftrazidime CA , Ampicillin A,
Azithromycin AT, Aztreonam AO , Carbenicillin CB, Ceftaxime CE, Cefotaxin
CN , Amoxicillin/Clavulanic acid AC ,Ceftriaxone CI,
Cifixime CFX,
Erythromycin E, Gentamicin G, Levofloxacin LE, Nitrofluranton NF, Pencillin G
P, Pipracillin PC, Tetracycline T, Trimethoprim TR and Vancomycin.
Therefore, AE36 and AE38 isolates were chosen to be used for further
experiments. The results indicated that the percentage of multi drug resistant S.
aureus isolates ( resistance to 12 or more antibiotics) was (88.4% ) (Appendix- 3),
and this was in agreement with the findings of Shittu and Johnson,(2006) who
Chapter three
Results and Discussion
79
noted that 88% of the isolates were multi-drug resistant ( resistance to three or
more antibiotics). The results demonstrated that, 41.8% were resistant to
Ciprofloxacin,
100%
to
Vancomycin
,100%
to
Ceftaxime
,100%
to
Amoxicillin/clavulanic acid and 69.7% to Tetracycline. Whereas Suo’d, (2005)
mentioned that the percentage of resistance to the same antimicrobial were ( 60 , 0,
71.4, 100, 60)% respectively, while Albaldawi ,(2005) reported in her study that
the resistance were ( 36.9, 21.1, 94.8, 36.9, 63.2 ) % respectively.
Moreover, the present study revealed that the resistance to Penicillin G and
Nitrofurantoin was 100% and this agrees with Suo’d, (2005) .In addition, the study
isolates demonstrated 46.5% resistance to Rifampin, but in Albaldawi , (2005), the
percentage was 15.6%, and 32.5% to Gentamicin that agreed with Albaldawi ,
(2005) which was 36.1%.
The differences in the level of susceptibility to certain antimicrobial and the
mechanism of resistance to antimicrobials could be attributed to the following;
· Reduced cell wall permeability and production of chromosomal and plasmid
mediated β- lactamase which is the principle mechanism of Methicillin
resistance .(Katzif et al ., 2005)
· Production of biofilm helped producing highly resistant organisms for killing
by bactericidal antimicrobials which is due to slow diffusion of antimicrobial
(Otto.,2006)
· Multidrug efflux systems( Raygada and Levine, 2009).
In addition , resistance of S. aureus to antibiotics is definitely associated with
over and randomly use of broad-spectrum antimicrobials in hospitals.
Chapter three
Results and Discussion
Figure (3-4)Susceptibility of S. aureus to antimicrobials
Amikacin AK
Ceftazidime CA
Amoxicillin/Clavulanic acid AC
Ceftriaxone CI
Nitrofurantoin NF
Ampicillin A
Cifixime CFX
Oxacillin OX
Azithromycin AT
Ciprofloxacin CF
Pencillin G P
Aztreonam AO
Erythromycin E
Pipracillin PC
Carbenicillin CB
Gentamicin G
Rifampin R
Ceftaxime CE
Imipenem I
Tetracycline T
Cefotaxin CN
Levofloxacin LE
Trimethoprim TR
Vancomycin VA
Methicillin M
80
Chapter three
Results and Discussion
81
3.5 Detection the ability of S.aureus isolates to produce
Staphyloxanthin
The results of identifying the ability of S.aureus isolates (43isolate) for
staphyloxanthin production
on different culture media are shown in Appendix
(4) and Figure (3-5) . It was found that milk agar medium was the best media for
staphyloxanthin production, hence the isolates percentage which gave the highest
orange and yellow pigment production were 72.1%. While, Trypticase yeast
medium and Trypticase soya medium revealed percentages 37.2%.Peanut seeds
medium and sesame seed medium appeared percentage 30.2 % for orange
staphyloxanthin production. No production of orange pigment were observed in
several media such as sunflower seeds medium, brain heart infusion agar,
carotinoid expression medium , muller Hinton Agar and nutrient agar . (Appendix 4)
These results suggested that specific nutrients are required for
staphyloxanthin production .According to the results pigment producer isolates
(AE32 , AE36 ) and non pigment producer isolate (AE38 ) were selected and used in
the subsequent experiments .
Medium components may be critical for production of staphyloxanthin .The fatty
acid as a carbon source is a better substrate for the growth of bacteria than sugars
.based on the comparison between the composition of different fatty acid
containing seeds and oils . The saturated form of fatty acid could be a better choice
of carbon source for the maximum production of pigment ( Giri et al ., 2004) .
Chapter three
Results and Discussion
82
Type of medium
Figure (3-5) Influence of different culture media on the production of staphyloxanthin
*Milk: Milk agar medium , TYATrypticase yeast medium ,TSA:Trypticase soya medium ,PNSA: Peanut seeds
medium , SSA:Sesame seed medium , SFSA: Sunflower seeds medium , BHIA: Brain-Heart Infusion Agar, N.A:
Nutrient Agar MHA: Mueller-Hinton Agar CEM: Carotinoid expression medium,
3.6 Extraction of Staphyloxanthin
Staphyloxanthin was extracted by using different solvents: acetone (Giri et
al.(2004); Godinho and Bhosle ,2008) , chloroform (Ra’aof and Latif,2010) , ethyl
acetate ( Anuradha et al. (2004); Kurjogi et al.,2010 ) and by methanol( Marshall
and Wilmoth ,1981b). The best results were obtained by using methanol, being
easier with a good amounts to (0.2 g/ml), table (3-4) and figure(3-6-a,b,c,d) .Our
result was in agreement with Chamberlain et al., (1991) who extracted the pigment
in a concentration of 0.2 mg /ml. The pigment suspension was exposed to several
wave lengths (300-600) nm using spectrophotometer. The results revealed three
Chapter three
Results and Discussion
83
peaks in the wave lengths ( 315,450,463) nm. This result was closely related to
some previous studies (Marshall and Willmoth,1981a;Wieland et al.,1994:
Lipovsky et al.,2008) in which high peak of pure pigment absorbance appeared at
the wavelength of (450) nm .
Table (3-4) Optical density of solvents
Solvents
Optical Density (OD) nm
Maximum Abservation
Methanol
450
450
Acetone
460
-
Chlorophorm
520
-
Athyl acetate
535
-
(-) No peak in diagram
Figure (3-6-a) Absorption curve of staphyloxanthin
Spectrophotometer.
extracted by using Ethyl acetate using UV-
Chapter three
Figure (3-6-b) Absorption curve of staphyloxanthin
Results and Discussion
84
extracted by Chloroform using UV-
Spectrophotometer
Figure (3-6-c) Absorption curve of staphyloxanthin
Spectrophotometer
extracted by Aceton using UV-
Chapter three
Results and Discussion
85
Figure (3-6-d) Absorption curve of staphyloxanthin
extracted by Methanol using UV-
Spectrophotometer
3.7 Identification of staphyloxanthin producing isolate
The best pigment producing isolate was identified by culturing the two isolates
AE36 and AE32 on milk agar at 37oC for two days then incubated at 28oc for two
days (Grinsted and Laccy, 1973) , the pigmentation was calculated quantitatively
according to Tao et al.,(2010). The AE36 isolate revealed higher pigmentation
(165.21) unit/cell ( cuvate volume 3ml ) compared with the pigmentation by AE32
which was (131.20) unit/cell (Figure 3-7). Therefore, the AE36 isolate was chosen
for further experiments
Chapter three
Results and Discussion
86
Figure (3-7) S. aureus staphyloxanthin production
3.8 Optimization the conditions for staphyloxanthin production
Statistical significant differences (p<0.05) were detected for the five experiments.
3.8.1 Effect of medium composition
Results showed that the culture media used in this study: milk agar ,nutrient agar,
Muller Hinton agar, carotenoid expression media, trypticase yeast agar and
trypticase soya agar were different in their ability to induce staphyloxanthin
production which may due to the difference in medium composition . It was found
that the best medium for pigment production was milk agar with an amount of
pigment produced for AE36 isolate (165.21) unit/cell (cuvate) compared with the
pigment produced in TSA and TYA, which was (89.22 , 77.22) unit / cell (cuvate)
respectively . In the CEM the production was less than 37.9 unit/cell (Figure 3-8
Chapter three
Results and Discussion
87
and Figure 3-9) and table (3-5). These results showed similarity with the results
mentioned by Grinsted and Lacy, (1973).
Type of medium
Figure (3-8) Staphyloxanthin production from S. aureus AE36 isolate grown in different media.
Each value represent the mean ± SD, where ( n=20) .The SD value = 0.06
A : Brain-Heart Infusion Agar
B : Carotinoid Expression Medium
C : Milk agar
D : Mueller-Hinton Agar
E : Nutrient Agar
F : Peanut seeds medium
G : Sunflower seeds medium
H : Sesame seed medium
I : Trypticase soya medium
J : Trypticase yeast medium
Chapter three
Results and Discussion
A
Peanut seed medium
Sesame seed medium
Sunflower seeds
B
88
Chapter three
Trypticase yeast
di
Nutrient Agar
Milk agar
Mueller-Hinton Agar
Results and Discussion
89
Chapter three
Results and Discussion
A
B
Carotinoid Expression
Medium
Brain-Heart Infusion
Agar
Trypticase soya
medium
Figure (3-9) Growth of S.aureus in different media for 72hr at 370C
A: AE38 White isolate
B: AE36 Orange isolate
90
Chapter three
Results and Discussion
91
The variation in the productivity of pigment production may be due to the
complexity of the medium component. Some simple media might be consumed
easily from the bacteria, and some being complicated find hard to profited from it.
Hence to obtain good amount of staphyloxanthin which is secondary metabolize.
(Krinsky, 1974 ;Fourmier and Philpott,2005). S. aureus secreted lipase enzymes
that breakdown the fatty acids and benefited from it as carbon and energy sources,
this substrate induced surfactants compound production such as carotenoids that
had metabolic control system similar to control system for staphyloxanthin (Mishra
et al., 2009). Therefore the surfactant compounds stimulated the production of
pigment (Figure 3-10), (Table 3-5). In the case of seven medium ( Milk medium ,
Peanut seeds medium, Sesame seed medium, Sunflower seeds medium, Brain-Heart
Infusion Agar, Mueller-Hinton Agar) the results showed significant differences ,
but in the case of Carotinoid Expression Medium and Nutrient Agar there was no
significant differences and the same case in resuls of Tripticase soya medium and
Tripticase yeast medium there was no significant differences .
Table (3-5) The quantity of staphyloxanthin in different media
Type of Media
Brain-Heart Infusion Agar
Carotinoid Expression
Medium
Milk agar
Mueller-Hinton Agar
Nutrient Agar
Peanut seeds medium
Sesame seed medium
Sunflower seeds medium
Tripticase soya medium
Tripticase yeast medium
Optical density at
(450) nm
0.393
0.891
Quantity of pigment
Unit/cell (cuvate)
16.6
37.9
1.96
0.628
0.902
1.559
1.290
0.566
1.807
1.88
165.21
26.6
38.4
66.5
55.06
24.03
77.22
89.22
Chapter three
Results and Discussion
92
3.8.2 Determination of optimum temperature to staphyloxanthin
production
The effect of different temperatures (20, 25, 28, 37, 40)oC on staphyloxanthin
production were examined. Figure (3-10) shows that the pigment production was
increased at 37oC, whereas decreased at the low temperatures (less than 20oC) and
in high temperature (more than 37oC) , the 37oC was the optimum temperature for
pigment production, and this result was similar to the results obtained by
Chamberlain et al.,(1991) and Kim and Lee,(2012). Whereas other studies found
that the optimum temperature for pigment production was ( 30oC ) (Kurjog et
al.,2010). Others studies found that the optimum temperature for pigment
production when grown in CEM was 30oC (Xiong and Kapral 1992) , whereas it
was 28-37oC when bacteria grown on milk agar (Grinsted and Lacey, 1973; Xiong
and Kapral 1992), while this bacteria can produce pigment in a temperature more
than 40oC when grown in medium contained oil seed such as sunflower seeds
medium, sesame seed medium and peanut seeds medium ( Giri et al .,2004).
Decreased of staphyloxanthin production at high temperatures had several
reasons:
-
High temperature inhibit the genes which are responsible for pigment
production expression without effecting the bacterial growth (Silva et al.,2012)
-
High temperature inhibit the enzymes responsible for the condensation of
pigment production (Staphyloxanthin Condensing Enzyme SCE) which is very
sensitive to high temperatures ( Pelz et al., 2005).,
The bacterial growth in high temperatures leads to loss the ability for producing
pigment as is a secondary metabolite which is not necessary for bacterial growth .
This may be the reason that some clinical isolates were pigmented because the
Chapter three
Results and Discussion
93
bacterial growth inside the human body is normally at 37oC which is the optimum
temperature for pigment production ( Plata et al.,2009 ; Kim and Lee,2012). The
results showed significant differences (p< 0.05).
Figure (3-10) Staphyloxanthin production from S. aureus AE36 isolate grown in different
temperatures. Each value represent the mean ± SD , where ( n=20) .The SD value = 5.90
3.8.3 Determination of optimum pH for pigment production
The effect of pH on the metabolic activity of bacterial cell and particularly
production for metabolites such as staphyloxanthin pigment was studied. Five
different pH values ( 5, 6 , 7, 8, 9) were examined to find the optimum pH for
Chapter three
Results and Discussion
94
pigment production . The result revealed that the optimum pH was 8 (Figure 3-11) ,
Generally the pigment production was higher in alkaline media, this agrees with
Wieland et al.,(1994) finding. This is because at pH 8 the activity of proline oxidase
enzyme is inhibited which cause anabolism of proline the basic amino acid for
pigment production , whereas lower or higher pH than 8 lead to imbalanced or
break in the biological pathway that lead to pigment production which affected by
the activity of the enzyme responsible for pigment production (Xiong and Kapral,
1992; Wieland et al., 1994). The results showed significant differences (p< 0.05).
Figure (3-11) Staphyloxanthin production from S. aureus AE36 isolate grown in different pH
values. Each value represent the mean ± SD , where ( n=20) .The SD value = 9.77
Chapter three
Results and Discussion
95
3.8.4 Determination of optimum incubation time for pigment
production
The optimum incubation time for pigment production was detected by
incubating bacteria in milk agar at five different times (18, 24, 36, 48, 72) hour,
(Figure 3-12). It was found that 72 hr was the optimum incubation time for pigment
production because the pigment is a secondary metabolite (Clauditz et
al.,2006;Kwiecinski et al.,2009) which required time to produce. After 24 hr of
incubation a few amount of pigment was appeared and the colonies became colored
with light yellow pigment, then the color was increased with the incubation time
until the pigment production reached the stationary phase after 72hr .The same
results mentioned by Clauditz et al.,(2006). The results showed significant
differences (p< 0.05).
Figure (3-12) Staphyloxanthin production from S. aureus AE36 isolate grown in milk agar at different
incubation periods. Each value represent the mean ± SD , where ( n=20) .The SD value = 8.575
Chapter three
Results and Discussion
96
3.8.5 Effect of aeration on staphyloxanthin production
The aeration effect on pigment production was studied using three volumes of
Milk medium (25, 50, 100) ml in 250 ml flask, incubated at 37o C for 72 hr in a
shaker. The results demonstrated that the higher production of pigment was
obtained when 50 ml medium was used (165.21) unit/cell, while the production
decreased when the volume of the media was more or less than 50 ml/flask (Figure
3-13).
When the volume of culture media was 25 ml the ratio of surface area to the
volume of media was high which provided high ratio of oxygen that made bacterial
growth higher than the pigment production because the pigment is a secondary
metabolite and is not necessary for bacterial growth (Mishra et al.,2011). In
addition the small volume of media do not provide the suitable amount of substance
which when consumed cause decreased in pigment production (Sun et al.,2012).
The pigment production decrease when the culture volume was increased because
of the surface area become smaller. This leads to decrease in oxygen ratio in the
culture ,and then decreased in pigmentation because the anaerobic condition
blocked the primary step of pigment production. In addition to inhibition into
condensation of farnesyl diphosphate molecules together to form pigment
(Hammond and White , 1970;Mishra et al.,2011; Sun et al.,2012). The results
showed significant differences (p< 0.05).
Chapter three
Results and Discussion
97
Figure (3-13) Staphyloxanthin production from S. aureus AE36 isolate grown in different volumes
of milk medium. Each value represent the mean ± SD, where ( n=20) .The SD value = 17.53
3.9 Purification of Staphyloxanthin
The thin layer chromatography (TLC) method was used to purify
staphyloxanthin. TLC is a general method used to purify secondary metabolism
products (Whitaker and Bernard,1972). The RF value was 0.38 (Figure 3-14).
Which was similar to the results of Marshall and Willmoth,(1981b) and Wieland et
al.,(1994) , with the Molecular Wight of staphyloxanthin is 819.17 dalton.
This small difference in the wave length (absorbance) of the purified pigment
suspension may be due to the compounds in the medium which was used or to the
methods of the extraction and purification . The results elucidate visible orange ,
yellow spots and invisible spots emerged under ultra violet light .
Results and Discussion
Distance of solvent
Chapter three
A
Distance of Staphyloxanthin
Other invisible composition of pigment
B
98
Other visible
composition
of pigment
The first position
Figure (3-14) Partial purification of Staphyloxanthin from S.aureus AE36 by using Thin Layer
Chromatography (Molecular Wight of staphyloxanthin is 819.17 dalton , mobile phase: benzenemethanol-acetic acid 87:11:2 (vol/vol/vol) )
A: Visible Spots.
B: Invisible Spots.
Chapter three
Results and Discussion
99
3.10 Anti bacterial activity of staphyloxanthin against bacteria
The antibacterial activity of the pigment was examined against several bacterial
geneses (Staphylococcus epidermidis, Pseudomonas aeruginosa, Salmonella spp.,
Shigella spp., Escherichia coli, Klebsiella spp , Proteus spp. Pseudomonas
fluorescens , Pseudomonas putida , Staphylococcus aureus isolate AE36,
Staphylococcus aureus isolate AE32 and Staphylococcus aureus isolate AE38) with
concentration 0.2 g /ml revealed that staphyloxanthin has no activity against tested
bacteria that used in this study ( Figure 3 – 15 ).This results were agreed with
earlier studied by Chamberlain et al.,( 1991), who reported the staphyloxanthin
pigment extract did not appeared antibacterial activities against several gram
positive and negative bacteria at
concentration 0.25 mg /ml, as well as
Samaranika, (2012), who confirmed the results of this study about the
staphyloxanthin pigment extract has no activity against Klebsiella spp.
Figure (3-15) Antibactirial activity of staphyloxanthin against Klebsiella spp by using well
diffusion method on MHA for 24hr at 370C
Chapter three
Results and Discussion
100
3.11 The induction of mutants with chemical agent
3.11.1 Mutation of bacterial isolate by Ethyl Methane Sulfonate
(EMS)
S. aureus AE36 isolate was treated with chemical mutagen EMS in order to
obtain mutants that differed from the wild type in terms of Pigment production ,
which was measured after the exposure to chemical mutagens.
The exposure
S. aureus AE36 with EMS (final concentration of 10 µg/ml )
showed the staphyloxanthin production was completely lost in the mutant one
( Figure 3- 16) .because EMS is a mutagen which produces random mutations in
genetic materials due to nucleotide substitution; particularly by guanine alkylation.
This typically produces only point mutations (Vijver et al.,1975).
B
A
Figure (3-16) Mutation of S.aureus AE36 on milk agar for 72hr at 370C
A: Control isolate AE36
Chapter three
Results and Discussion
101
B: AE 36 mutated using ethyl methane sulphonate.
3.12 Experimentally infected mice with bacteria and treatment with
antimicrobial ointment
The scarified animals at their back were infected with Staphylococcus aureus
AE36 , AE38 and with the mutant ∆AE36. The treatment was achieved by
Ciprofloxacin ointment (5 µg / ml) for three times per day for seven days . After 4
days of treatment the results showed that group A animals( control normal saline)
were still alive and no infection occurred (Figure 3-17-a) (Table 3-6) . This means
that the normal saline used was not contaminated .
Table (3-6) Infection period of mice by S. aureus
Group
Infection Treatment Viable count
Viable count (CFU/ml)of
days
(CFU/ml)of
bacteria after appearance
bacterial
of infection
days
inoculum before
inoculation
Group A
4
-
-
-
Group B
4
5
1.7 × 107
2.5 × 109
Group C
4
5
1.7 × 107
7 × 107
Group D
5
6
1.7 × 107
1.7 × 108
*Group A inoculated by normal saline
*Group C inoculated by AE38
* Group B inoculated by AE36
*Group D inoculated by ∆AE36
Chapter three
Results and Discussion
102
Group B animals (infected with AE36) were all infected after 4 days from
receiving the inoculum and all were treated for 5 days , thus the totally healing
occurred after 10 days from infection ( Figure 3-17-b) and (Table 3-6).
Group C animals (infected mice with AE38) were all infected after 4 days from
receiving the inoculum . However, the intensity of infection was milder than that in
group B animals, thus the totally healing occurred after 8 days from infection
(Figure 3-17-c) and (Table 3-6).
Group D animals (infected mice with AE36 mutant) were all infected after 5 days
from receiving inoculums, but the intensity of the infection was less than in group B
and more than group C. This result is probably because of the pigmentation in this
group was lost as well as it’s virulence ability. This explained that the STX can be
considered as a virulence factor but without antibacterial effect . Hence the risk of
being infected with pigmented S. aureus is higher than that from the non pigmented
ones(Figure 3-17-d) and (Table 3-6).
Chapter three
( A ) Mice received Saline.
Results and Discussion
103
( B ) Mice received (1×107) CFU/ml
S. aureus yellow pigment AE3.
( C ) Mice received (1×107) CFU/ml S.
( D ) Mice received (1×107) CFU/ ml S. aureus
aureus white pigment production AE38.
mutant of pigmentation (mutant ∆AE36).
Figure (3-17) Mice infected with A: normal saline, B: AE36, C: AE38, D: ∆AE36
Chapter three
Results and Discussion
104
3.13 Staphyloxanthin as anti oxidant factor with human whole blood
cell and neutrophil
After extraction and partial purification of staphyloxanthin , it was tested as anti
oxidant with human whole blood cell . The pigmented isolate AE36, non pigmented
isolate AE38 and mutant one from isolate AE36 . The results showed that the
neutrophil count was decreased as in the sample B (Figure 3-18) and (Table 3-7a,b), and increased in sample C and D.These results showed that when the amount
of pigment was increased the neutrophil count was decreased, this is because the
pigment production is related with the free oxygen.
The yellow-orange pigment produced by most clinical isolates of Staphylococcus
aureus has been associated with enhanced bacterial survival in harsh environments
and increased staphylococcal pathogenicity , pigment production by S. aureus has
been tied to bacterial virulence by the finding that it impairs the antimicrobial
action of neutrophils (Liu et al.,2005). In the first case there were no significant
differences (p< 0.05), but in the second case there were significant differences (p<
0.05).
Table (3-7-a) Staphyloxanthin as anti oxidant factor with healthy individual whole blood cell and
neutrophil count
Leucocytes
Control Blood
(A)
Neutrophils
59%
AE36 (B)
16%
with Blood with
AE38 (C)
46%
Blood with mutant
AE36 (D)
40%
Chapter three
Results and Discussion
105
Table (3-7-b) Staphyloxanthin as anti oxidant factor with allergic individual whole blood cell and
neutrophil count
Leucocytes
Neutrophils
Control Blood with AE36 Blood
with Blood with mutant
(A)
(B)
AE38 (C)
AE36 (D)
72%
29%
35%
31%
Figure (3-18) Human whole blood cell count and neutrophil (100 X)
A: Normal blood count with PBS.
B: Blood with S. aureus AE36.
C: Blood with S. aureus AE38.
D: Blood with mutant S. aureus AE36.
Chapter three
Results and Discussion
106
3.14 Molecular diagnostic method for detection of Staphyloxanthin
pigment by Real-Time PCR
3.14.1 DNA Extraction
The chromosomal DNA was extracted from bacterial cell by using two methods:.
3.14.1.1 Salting out method
This method was used to extract the total chromosomal DNA by using organic
solvent (break the cell wall) and enzymes.
The DNA from three selective isolates was extracted efficiently by using salting
out method, as shown in Figure (3-19). The yield of the DNA extracted from
bacterial isolates were ( 3345, 2790, 5700 ) µg/ml with purity of (1.9, 1.6, 1.8 ),
for AE36, AE38, ∆AE36 respectively ( Table 3-8 ) .
Table (3- 8) The purity and concentration data of total DNA extracted from
selected isolates of S.aureus
Isolates No.
A260/A280 ratio
Yield : µg/ml
AE38
1.6
2790
AE36
1.9
3345
AE32
1.7
2950
AE23
1.8
3975
DAE36
1.8
5700
Chapter three
Results and Discussion
1
2
3
4
107
5
Chromosomal DNA
Plasmid DNA
Figure (3-19 ) Agarose gel electrophoresis of DNA extracted by using salting out method from
S.aureus isolates by electrophoresis on a 0.8 % agarose gel (voltage 5volt/cm, during 1.5hr)and
visualized under UV light after staining with ethidium bromide.
Lane 1 : DNA extract from AE36 isolate
Lane 2 * : DNA extract from AE32
Lane 3* :DNA extract from AE23
Lane 4 :DNA extract from ∆ AE36
Lane 5 :DNA extract from AE38
* This chromosomal DNA lane do not used in this study
Chapter three
Results and Discussion
108
3.14.2 Detection of Staphyloxanthin pigment and MRSA .
The RT-PCR assay was achieved using three selected isolates , S. aureus AE36
and S. aureus AE32 ( methicillin resistant -staphyloxanthin pigment production)
and S. aureus AE38 ( non pigment producer and susceptible to methicillin ) by
using SYBR Green RT- PCR Assay. The results are shown in Figures ( 3-20 , 3-21
and 3-24). Two curves were seen for S. aureus AE36 and S. aureus AE32 (positive
result), and two lines under the threshold line appeared indicating the negative
result (AE38 , Negative control)( Figures 3-22 , 3-23 3-25 ),
Figure (3- 20) : The Real Time - PCR run for the AE36 isolate by SYBR Green RT- PCR
Assay.
Chapter three
Results and Discussion
109
Figure (3- 21) : The Real Time - PCR run for the AE32 isolate by SYBR Green RT- PCR
Assay.
Figure (3- 22) : The Real Time - PCR run for the AE38 isolate by SYBR Green RT- PCR
Assay.
Chapter three
Results and Discussion
Figure (3- 23) : The Real Time - PCR run by SYBR Green RT- PCR Assay.
110
( negative
control).
AE36
AE32
Figure (3-24) : The SYBR Green RT- PCR Assay, the positive result for AE36 isolate
(first curve ) and the result for AE32 appear as a second curve .
Chapter three
Results and Discussion
111
AE38
Negative
control
Figure (3-25) : SYBR Green RT- PCR Assay, the negative result for AE38 isolate (curve
one) and negative control appear as a line under the threshold line(negative result) .
This
study
determines
the
methicillin
-
resistant
Staphylococcus
aureus (MRSA) ,the detection method based on the melting temperature analysis
profiling of S. aureus clinical isolates from blood. Duplex real-time PCR assay
was used for the simultaneous detection of crtM ( staphyloxanthin pigment
production) and mecA (methicillin-resistance) genes in a single SYBR Green I
real-time PCR tube assay. Evaluations were based on the melting temperature (T m)
analysis of the amplicons using S. aureus clinical isolates (Real-time PCR
amplification products with melting peaks at 80 ± 2°C) each Real-time PCR assay
was completed within two hours. This rapid genotypic method is useful for the
detection of resistant determinant (mecA) and identification of S. aureus (crtM)
staphyloxanthin pigment production isolates.
Methicillin resistance in S. aureus is caused by the acquisition of an exogenous
gene, mecA, that encodes an additional β-lactam-resistant penicillin-binding
protein (PBP), (Huletsky et al.,2004). The assay involves several basic steps,
namely, DNA extraction, amplification by thermal cycling, and amplicon
Chapter three
Results and Discussion
112
detection. The pigment produced by Staphylococcus aureus is the deep-yellow
carotenoid 4,4'-diaponeurosporene after prolonged cultivation, this pigment is in
part converted to the orange end product staphyloxanthin. DNA sequencing of this
fragment revealed two open reading frames (ORFs) which are very likely
cotranscribed. ORF1 encodes a 254-amino-acid hydrophobic protein, (CrtM).
ORF2 encodes a 448-amino-acid hydrophobic protein, (CrtN), Spectrophotometry
and gas chromatography-mass spectrometry analyses of the carotenoid production
of E. coli and S. aureus clones containing either ORF1 or both ORFs together
suggest that ORF1 and ORF2 represent the dehydrosqualene synthase gene (crtM)
and the dehydrosqualene desaturase gene (crtN), respectively (Wieland et al
.,1994)..
Lan et al.,(2010) reported that golden pigmentation of S. aureus is the product of
a C30 triterpenoid carotenoid biosynthesis pathway, and the carotenoid pigment
biosynthesis genes are organized in an operon crtOPQMN controlled by the
alternative sigma factor SigB ,and hypothesized that genes affecting pigmentation
may also influence the production of virulence determinants and have an impact on
the pathogenesis of S. aureus.
The amount of DNA molecules was monitored during the course of the
reaction by recording the amount of fluorescent light emitted which achieved by
adding fluorescently labeled DNA dyes into the PCR reaction. In real-time PCR
the amplified product is detected via fluorescent dyes. These are usually linked to
oligonucleotide which bind specifically to the amplified product . Monitoring the
fluorescence intensities during the PCR run allows the detection and quantitation
of the accumulating product without having to re-open the reaction tubes after the
PCR run (Mackay, 2004)
۱۱۳
Conclusions and Recommendations
1- The percentage of Staphyloxanthin production of S.aureus isolates
was estimated 72.1% .
2- The
best
method
for
extraction
of
partial
purification
staphyloxanthin was by using Methanol.
3- The optimum conditions for Staphyloxanthin production were at pH
-8, 370C , and for a 72 hr. The milk agar medium revealed the
highest production of pigment .
4- Staphyloxanthin is a virulent factor and antioxidant factor.
5- The results revealed
that the staphyloxanthin has no antibacterial
effect against the tested bacteria using in this study.
6- The
use of rapid and powerful Real-Time PCR assay appeared
useful for detection of mecA gene (MRSA) and crtM gene( pigment
production).
Conclusions and Recommendations
114
1. Studying the role of quorum sensing in production of virulence factors
from Staphylococcus aureus especially Staphyloxanthin pigment.
2. The study of the gene expression of the operon responsible for the
carotenoid pigment production of Staphylococcus aureus
3. Immunological studies and its applications of Staphylococcus aureus
staphyloxanthin .
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III
List of Contents
Item
Subject
Page
no.
Summary
Ⅰ
Subject Index
Ⅲ
Table Index
Ⅺ
Figure Index
XIII
List of Abbreviations
XV
Introduction
1
Chapter one / Literature Review
1.1
General description of staphylococcacea
4
1.2
Classification of Staphylococcus aureus
4
1.3
General characteristics of S. aureus
5
1.3.1
Staphylococcus aureus subsp. aureus
5
1.3.2
Staphylococcus aureus subsp. Anaerobius
6
1.4
Pathogenicity.
6
1.5
Virulence factors of S. aureus
7
1.5.1
Enzymes
10
1.5.2
Toxins
10
1.5.2.1
Superantigens
10
1.5.2.2
Exfoliative toxins
11
1.5.2.3
Other toxins
11
IV
1.5.3
Other immunoevasive strategies
11
1.5.3.1
Protein A
11
1.5.3.2
Staphylococcal pigment ( Staphyloxanthin)
12
1.6
Antibiotic resistance of Staphylococcus aureus
14
1.7
Characteristic of Staphyloxanthin
19
1.8
Structure and biosynthesis of staphyloxanthin
20
1.9
Optimization of conditions for staphyloxanthin
production
22
1.9.1
Effect of medium composition
22
1.9.2
Effect of temperature
23
1.9.3
Effect of pH
23
1.9.4
Effect of incubation time
23
1.9.5
Effect of aeration
23
1.10
Extraction and purification of staphyloxanthin
24
1.10.1
Extraction of staphyloxanthin
24
1.10.2
Thin Layer Chromatography
24
1.11
Genetic expression of staphyloxanthin
25
1.12
The crt operon
26
1.13
Mutation of staphyloxanthin
27
1.14
Regulated promoter of the crt operon from S. aureus
27
1.15
Real Time PCR technique
28
Chapter two /Materials and Methods
2.1
Materials
30
V
2.1.1
Instruments and equipments
30
2.1.2
Chemicals and biological compounds
31
2.1.3
Enzymes
34
2.1.4
Other materials
34
2.1.5
Culture media
35
2.1.5.1
Ready-made media
35
2.1.5.2
Prepared culture media
36
2.1.5.2.1
Blood agar
37
2.1.5.2.2
Gelatin agar medium
37
2.1.5.2.3
Deoxyribo nuclease (DNAse) media
37
2.1.5.2.4
Milk agar medium
37
2.1.5.2.5
Peanut seeds medium
37
2.1.5.2.6
Sunflower seed medium
38
2.1.5.2.7
Sesame seed medium
38
2.1.5.2.8
Triypticase yeast medium
38
2.1.5.2.9
Carotinoid expression medium
38
2.1.5.2.10
Triypticase soya medium
38
2.1.5.2.11
Carbohydrate fermentation medium
39
2.1.5.2.12
Milk broth medium
39
2.1.6
Analytical profile index ( API System)
39
2.1.7
Antimicrobials
39
2.1.7.1
Antimicrobial Disks
39
2.1.7.2
Antimicrobial ointment
41
VI
2.1.8
Bacterial isolates
41
2.1.9
Solutions and reagents
42
2.1.9.1
Gram solutions
42
2.1.9.2
Oxidase test reagent
42
2.1.9.3
Catalase reagent (3%)
42
2.1.9.4
Normal saline solution
42
2.1.9.5
Coagulase test
42
2.1 .9.6
Stock salt solution
42
2.1.9.7
Sodium thiosulphate (5 %)
43
2.1.9.8
McFarland solution (tube No. 0.5)
43
2.1.9.9
Phosphate Buffered Saline (PBS)
43
2.1.9.10
Sorensen’s Phosphate buffer (Leishman stain buffer)
44
2.1.9.11
Solution for mobile phase of thin layer
chromatography
44
2.1.9.12
Seeds extract
44
2.1.10
Solutions for DNA extraction
44
2.1.10.1
Tri-ethylene diamine tetra acetic acid (TE)buffer
44
2.1.10.2
Tris- boric acid- ethylene diamine tetra acetic acid
(TBE) buffer
44
2.1.10.3
Sodium dodesyl sulfate solution (SDS 10%)
45
2.1.10.4
Lysozyme solution
45
2.1.10.5
Ethidium bromide
45
2.1.10.6
Sodium chloride solution (5M)
45
2.1.10.7
Saline EDTA Tris Hcl (SET) buffer
45
VII
2.1.10.8
Loading buffer
46
2.1.10.9
Agarose gel ( 0.8%)
46
2.1.11
Laboratory animals
46
2.2
Methods
46
2.2.1
Collection of specimens
46
2.2.2
Characterization of specimens
47
2.2.3
Bacterial isolation
48
2.2.3.1
Cultural characteristics
48
2.2.3.2
Microscopic examination
48
2.2.3.3
Biochemical tests
48
2.2.3.3.1
Oxidase test
48
2.2.3.3.2
Catalase test
48
2.2.3.3.3
Coagulase test
49
2.2.3.3.3.*
Slid Coagulase test (Slid method)
49
2.2.3.3.3.** Coagulase test (tube method)
49
2.2.3.3.4
Blood hemolysis test
49
2.2.3.3.5
Urease test
50
2.2.3.3.6
Degradation of nucleic acid
50
2.2.3.3.7
Protease production
50
2.2.3.3.8
Gelatinase hydrolysis test
50
2.2.3.3.9
Sugar fermentation test
50
2.2.3.4
The characterization of the isolates using Api staph.
51
2.2.3.4.1
Preparation of the inoculums
51
VIII
2.2.3.4.2
Preparation of the strip
51
2.2.3.4.3
Addition of reagents
51
2.2.3.4.4
Reading results
52
2.2.4
Maintenance of bacterial isolates
52
2.2.4.1
Short-term storage
52
2.2.4.2
Medium-term storage
52
2.2.4.3
Long-term storage
52
2.2.5
Viable count determination of the bacterial inoculums
53
2.2.6
Susceptibility test for antimicrobials
54
2.2.6.1
Preparation of culture medium
54
2.2.6.2
Preparation of the inoculum
54
2.2.6.3
Inoculation
54
2.2.6.4
Application of the disks
54
2.2.6.5
Reading the results
55
2.2.7
Detecting the ability of S. aureus isolate to produce
Staphyloxanthin
55
2.2.8
Extraction of pigment staphyloxanthin (STX)
56
2.2.8.1
Extraction by acetone
56
2.2.8.2
Extraction by chloroform
57
2.2.8.3
Extraction by ethyl acetate
58
2.2.8.4
Extraction by methanol
58
2.2.9
Staphyloxanthin pigment assay
59
2.2.10
Determination of optimal conditions for
staphyloxanthin production
60
IX
2.2.10.1
Effect of medium composition
60
2.2.10.2
Determination of optimum temperature to
staphyloxanthin production
60
2.2.10.3
Determination of optimum pH for pigment production
61
2.2.10.4
Determination of optimum incubation time for
pigment production
61
2.2.10.5
Effect of aeration in staphyloxanthin production
61
2.2.11
Partial purification of Staphyloxanthin by thin layer
chromatography
62
2.2.12
Staphyloxanthin as antibacterial agent
63
2.2.13
The induction of mutants with chemical agents
63
2.2.13.1
Mutation by ethyl methane sulfonate (EMS)
63
2.2.14
The use of staphyloxanthin pigment as anti oxidant
with whole blood cell and neutrophils invitro
64
2.2.15
Study the effect of S. aureus (pigmented, non
pigmented and mutant ) isolates and treatment in an
animal model
65
2.2.15.1
Preparation of the bacterial suspension
65
2.2.15.2
Laboratory animals used
65
2.2.15.3
Experimentally infection mice with bacteria and
treatment with ciprofloxacin
66
2.2.16
Extraction of Bacterial DNA
67
2.2.16.1
Extraction by salting out
67
2.2.17
Estimation of the DNA concentration by the
Spectrophotometer
68
2.2.18
Agarose gel electrophoresis
68
X
2.2.19
Real –Time PCR technique
69
2.2.19.1
SYBR Green Real –Time PCR Assay
70
2.2.19.2
The procedure
70
2.2.20
Statistical anaysis
71
3- chapter three / Results & Discussion
3.1
Identification of Staphylococcus aureus
73
3.2
Standard curve of Staphylococcus aureus
76
3.3
Api staph test
77
3.4
Antimicrobial susceptibility of S. aureus
78
3.5
Detecting the ability of S. aureus isolate to produce
Staphyloxanthin
81
3.6
Extraction of staphyloxanthin
82
3.7
Identification of staphyloxanthin producing isolate
85
3.8
Optimization of conditions for staphyloxanthin
production
86
3.8.1
Effect of medium composition
86
3.8.2
Determination of optimum temperature to
staphyloxanthin production
92
3.8.3
Determination of optimum pH for pigment production
93
3.8.4
Determination of optimum incubation time for
pigment production
95
3.8.5
Effect of aeration on staphyloxanthin production
96
3.9
Purification of staphyloxanthin
97
3.10
Antibacterial activity of staphyloxanthin against
bacteria
99
XI
3.11
The induction of mutants with chemical agents
100
3.11.1
Mutation of bacterial isolate by Ethyl Methane
Sulfonate (EMS)
100
3.12
Experimentally infected mice with bacteria and
treatment with antimicrobial ointment
101
3.13
Staphyloxanthin as anti oxidant factor with human
whole blood cell and neutrophil
104
3.14
Molecular diagnostic method for detection of
staphyloxanthin pigment by Real-Time PCR
106
3.14.1
DNA extraction
106
3.14.1.1
Salting out method
106
3.14.2
Detection of Staphyloxanthin pigment and MRSA
108
Conclusions & Recommendations
Conclusions
113
Recommendations
114
References
115
Appendix
List of Tables
Table
no.
Title
1-1
Staphylococcus aureus virulence mechanisms
9
2-1
The instruments and equipments used
30
2-2
Chemicals and biological compounds used
31
Page no.
XII
2-3
List of Enzymes used in the study
34
2-4
Other materials were used
35
2-5
List of the diagnostic media used
36
2-6
The antimicrobial disks used in the study
40
2-7
The antimicrobial ointment used
41
2-8
The bacterial isolates were used throughout the study
41
2-9
Clinical samples collected from patients with
different infections
47
2-10
Reaction setup in R-T PCR
72
2-11
The program used in applied bio systems Real –Time
7500 PCR system
72
3-1
The biochemical tests of staphylococcus aureus
74
3-2
Frequency of S. aureus according to the site of
infection
75
3-3
Results of Api20-Staph for S.aureus AE36 isolate
77
3-4
Optical density of solvents
83
3-5
The quantity of staphyloxanthin in different media
91
3-6
Infection period of mice by S. aureus
101
3-7-a
Staphyloxanthin as anti oxidant factor with healthy
individual whole blood cell and neutrophil
104
3-7-b
Staphyloxanthin as anti oxidant factor with allergic
individual whole blood cell and neutrophil
105
3-8
The purity and concentration data of total DNA
106
extracted from selected isolates of S.aureus
XIII
List of Figures
Figure
no.
Title
Page
no.
1-1
Parts of the body and illnesses caused by S. aureus
7
1-2
S. aureus virulence factors
8
1-3
Proposed staphyloxanthin biosynthesis pathway
13
1-4
Biosynthetic pathway of carotenoid (STX) production
and comparative model of its effect on susceptibility
versus resistance to host defense CAPs in S. aureus
18
1-5
Structure of Staphyloxanthin
20
1-6
Organization of the staphyloxanthin biosynthesis genes
of S. aureus
26
1-7
crt operon and its expression
28
3-1
A standard curve of S.aureus AE36 isolate showing the
relationship between the optical density and the viable
bacterial count/ ml.
67
3-2
A standard curve of S.aureus AE38 isolate showing the
relationship between the optical density and the viable
bacterial count/ ml.
77
3-3
Api-20 Staph for identification of S. aureus
78
(AE36isolate)
3-4
Susceptibility of S. aureus to Antimicrobial
80
3-5
Influence of different culture media on the production
of staphyloxanthin
82
3-6-a
Absorption curve of staphyloxanthin extracted by
using ethyl acetate using UV-Spectrophotometer.
83
XIV
3-6-b
Absorption curve of staphyloxanthin extracted by
chloroform using UV-Spectrophotometer
84
3-6-c
Absorption curve of staphyloxanthin extracted by
acetone using UV-Spectrophotometer
84
3-6-d
Absorption curve of staphyloxanthin extracted by
methanol using UV-Spectrophotometer
85
3-7
S. aureus staphyloxanthin production
86
3-8
Staphyloxanthin production from S. aureus AE36 isolate
grown in different media
87
3-9
Growth of S.aureus on different media for 72hr at 370C
90
3-10
Staphyloxanthin production from S. aureus AE36 isolate
grown in different temperatures
93
3-11
Staphyloxanthin production from S. aureus AE36 isolate
grown in different pH values
94
3-12
Staphyloxanthin production from S. aureus AE36 isolate
grown in milk agar at different incubation periods
95
3-13
Staphyloxanthin production from S. aureus AE36 isolate
grown in different volumes of milk medium
97
3-14
Partial purification of staphyloxanthin from S.aureus
AE36 by using thin layer chromatography
98
3-15
Antibacterial activity of staphyloxanthin against
different bacterial geneses by using well diffusion
method
99
3-16
Mutation of S.aureus AE36 in milk agar for 72hr at 370C
100
3-17
Mice infected with: :A normal saline, B: AE36 , C: AE38
, D : ∆AE36
103
3-18
Human whole blood cell count and neutrophil
105
XV
3-19
Agarose gel electrophoresis of DNA extracted by using
salting out method from S.aureus isolates by
electrophoresis on a 0.8% agarose gel ,voltage
5volt/cm, during 1.5hr)and visualized by ethidium
bromide staining
107
3-20
The Real Time - PCR run for the AE36 isolate by
SYBR Green RT- PCR Assay
108
3-21
The Real Time - PCR run for the AE32 isolate by
SYBR Green RT- PCR Assay
109
3-22
The Real Time - PCR run for the AE38 isolate by
SYBR Green RT- PCR Assay.
109
3-23
. The Real Time - PCR run by SYBR Green RT- PCR
Assay. ( negative control).
110
3-24
The SYBR Green RT- PCR Assay, the positive result
for AE36 isolate (first curve ) and the result for AE32
appear as a (second curve)
110
3-25
SYBR Green RT- PCR Assay, the negative result for
AE38 isolate (one curve ) and negative control appear as
a line under the threshold line(negative result) .
111
List of Abbreviations
∆crtM
Mutant Carotenoid relative tail Moment gene
ANOVA
Analysis of Variance
ANTC4'IA
Aminoglycoside Adenyl Transferase 4' IA
Api 20 Staph
Analytical Profile Index Staph
BHI Agar
Brain heart infusion agar
BHI broth
Brain heart infusion broth
XVI
CA-MRSA
Community Associated Methicillin Resistant Staphylococcus
aureus
Cat
Chloramphenicol transferase
CEM
Carotenoid Expression Media
CFU/ml
Colony Forming Unit per milliliter
DNA
Deoxy ribonucleic acid
DNases
Deoxyribonuclease
DW
Distilled Water
EDTA
Ethylene Diamine Tetraacetic Acid
EMS
Ethyl Methane Sulphonate
ET
Exfoliative Toxin
MHA
Mueller-Hinton Agar
MRSA
Methicillin Resistant Staphylococcus aureus
MSA
Mannitol Salt Agar
MSCRAMM
Microbial Surface Components Recognizing Adhesive
Matrix Molecules.
MSSA
Methicillin Susceptible Staphylococcus aureus
NIT
Nitrate reduction
NCCLS
National Committee for Clinical Laboratory Standards
NCDCR
National Center for Drug Control and Research
OD
Optical Density
PBP
Penicillin Binding Protein
PBS
Phosphate Buffer Saline
XVII
PSI
pound per squared inch
PTSAgs
Pyrogenic toxin superantigens
PVL
Panton Valentine Leukocidin
RF
Relative Flow
ROS
Reaction oxygen species
RNA
Ribonucleic Acid
RNase
Ribo Nuclease
ROS
Reactive oxygen species
R-T PCR
Real – Time Polymerase Chain Reaction
Sig B
Sigma B gene
SCCmec
Staphylococcal Cassette Chromosome mec
SDS
Sodium Dodecyle Sulfate
SD
Standard Deviation
SEA-G
Staphylococcal enterotoxin A to G
SET
Saline EDTA Tris HCL
SPSS
Statistical Package for Social Science
SSSS
Staphylococcal Scalded Skin Syndrom
STX
Staphyloxanthin
TBE
Tris Boric acid –Ethylene Diamine Tetraacetic Acid
TE
Tris- Ethylene Diamine Tetraacetic Acid
TLC
Thin Layer Chromatography
Tm
Melting Temperature
XVIII
TSA
Trypticase Soya Agar
TSB
Trypticase Soya Broth
TSS
Toxic Shock Syndrome
TSST
Toxic Shock Syndrome Toxin
UTI
Urinary Tract Infection
UV
Ultra Violet
VP
Voges Proskauer
VRSA
Vancomycin Resistant Staphylococcus aureus
WT
Wild Type
APPENDIX
Appendix ( 1 ) Api Staph diagnostic tests
Reaction
Symbol
Negative result
Positive result
No substrate
0
Red
-
D-glucose
GLU
Red
Yellow
D-fructose
FRU
Red
Yellow
D-mannose
MNE
Red
Yellow
D-maltose
MAL
Red
Yellow
D-lactose
LAC
Red
Yellow
D-trehalose
TRE
Red
Yellow
D-mannitol
MAN
Red
Yellow
xylitol
XLT
Red
Yellow
D-melibiose
MEL
Red
Yellow
potassium nitrate
NIT
Colorless-light
red
pink
ß-naphthyl phosphate
PAL
yellow
violet
sodium pyruvate
VP
Colorless-light
violet-pink
pink
D-raffinose
RAF
Red
Yellow
D-xylose
XYL
Red
Yellow
D-saccharose
SAC
Red
Yellow
MDG
Red
Yellow
N-acetyl-glucosamine
NAG
Red
yellow
L-arginine
ADH
Yellow
orange-red
Urea
URE
Yellow
red-violet
(sucrose)
methylαDglucopyranoside
APPENDIX
Appendix ( 2 ) Antimicrobial disks used in the study with their diameter of
inhibition zone
Antimicrobial
Sym
Concentrati
agents
bol
on µg/disk
Amikacin
AK
Amoxicillin/cl
Diameters of inhibition zones
Resistant
Intermediate
30
14
15-16
17
AC
20/10
13
14-17
18
Ampicillin
A
10
13
14-16
17
Azithromycin
AT
15
13
14-17
18
Aztreonam
AO
30
15
16-21
22
Carbenicillin
CB
100
19
20-22
23
Ceftaxime
CE
30
14
15-22
23
Cefotaxin
CN
30
14
15-17
18
Ceftazidime
CA
30
14
15-17
18
Ceftriaxone
CI
30
13
14-20
21
Cifixime
CFX
5
15
16-18
19
Ciprofloxacin
CF
5
15
16-20
21
Erythromycin
E
15
13
14-22
23
Gentamicin
G
10
12
13-14
15
Imipenem
I
10
13
14-15
16
Levofloxacin
LE
5
15
16-18
19
Methicillin
M
5
9
10-13
14
Susceptible
avulanic acid
(mm)
APPENDIX
Continued
Nitrofurantoin
NF
300
14
15-16
17
oxacillin
OX
1
10
11-12
13
Pencillin G
P
10 unit
27
-
28
Pipracillin
PC
100
17
18-20
21
Rifampin
R
5
16
17-19
20
Tetracycline
T
30
14
15-18
19
Trimethoprim
TR
5
10
11-15
16
Vancomycin
VA
30
>15
-
15
APPENDIX
APPENDIX
Appendix ( 3 ) Antimicrobial susceptibility testing for 43 isolates of S. aureus
Isolates
code
AE 1
AE 2
AE 3
AE 4
AE 5
AE 6
AE 7
AE 8
AE 9
AE 10
AE 11
AE 12
AE 13
AE 14
AE 15
AE 16
AE 17
AE 18
AE19
AE 20
AE 21
AE22
AE23
AE24
AE25
AE26
AE27
AE28
AE29
AE30
AE31
AE32
AE33
AE34
AE35
AE36
AE37
AE38
Antibiotics
AK AC A AT AO CB CE CN CA CI CFX CF
I
R
R
R
R
I
R
R
R
R
I
I
S
R
R
R
R
R
R
I
R
R
R
I
R
I
R
I
R
R
R
R
R
S
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
S
R
R
R
I
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
S
R
R
R
R
R
R
R
R
R
S
R
R
I
R
R
I
R
R
S
S
S
R
R
R
R
R
R
I
R
S
S
S
S
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
S
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
S
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
I
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
I
S
R
R
R
S
R
I
I
S
R
R
S
S
S
S
R
R
R
R
R
R
R
S
R
R
S
R
S
S
S
R
S
S
S
S
S
S
E G
I LE M NF OX
P PC R
T
R
R
I
R
R
R
R
R
I
R
R
R
I
R
R
R
R
R
R
I
R
I
R
I
R
R
R
R
R
I
I
R
I
I
I
I
I
R
S
S
S
S
S
R
S
R
S
S
S
R
S
S
S
S
R
R
S
R
S
R
S
S
R
R
S
S
R
S
S
R
S
S
S
S
S
S
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
I
R
R
I
R
I
R
R
R
R
R
S
I
I
R
R
R
I
R
I
R
R
R
R
R
R
R
R
R
R
R
R
R
I
I
I
R
R
S
S
R
S
I
I
R
S
R
I
R
S
R
S
S
R
R
S
R
S
S
S
S
S
S
S
R
S
S
S
S
S
S
S
S
S
R
R
S
S
R
R
R
R
S
R
R
S
R
S
S
R
S
R
S
S
S
S
S
S
R
R
R
R
R
R
S
R
S
S
I
R
S
S
R
R
R
R
R
R
R
R
I
R
R
R
R
R
R
I
R
R
S
I
R
R
R
R
S
R
S
R
R
R
R
R
R
S
S
I
R
R
S
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
S
R
R
R
R
R
R
R
R
R
S
R
R
R
R
R
I
R
S
R
R
R
I
I
R
S
I
S
R
R
S
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
S
S
S
S
S
R
S
R
R
R
S
R
S
S
S
S
R
S
S
R
S
S
S
S
R
R
R
R
R
R
R
R
S
R
I
S
S
S
TR V
R
S
S
R
S
I
R
S
S
I
R
R
S
R
S
S
R
R
S
R
R
R
R
S
S
S
R
I
R
R
S
R
S
S
S
S
S
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
APPENDIX
AE39
AE40
AE41
AE42
AE43
R
R
R
I
R
R
R
R
R
R
R
R
R
R
R
I
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
S I
S R
S R
S I
R R
S R
R S
R S
S S
R S
R
R
R
S
R
S
R
R
I
R
R
R
R
R
R
S
R
R
I
R
R
R
R
R
R
R
R
R
R
R
R I
R R
S R
R I
R R
Amikacin (AK), Amoxicillin/Clavulanic acid (AC), Ampicillin (A), Azithromycin (AT), Aztreonam (),
Carbenicillin (CB), Ceftaxime (CE), Cefotaxin (CN), Ceftazidime (CA), Ceftriaxone (CI), Cifixime (CFX),
Ciprofloxacin (CF), Erythromycin (E), Gentamicin (G), Imipenem (I), Levofloxacin (LE), Methacillin (M),
Nitrofurantoin (NF), Oxacillin OX, Pencillin G (P), Pipracillin (PC), Rifampin (R), Tetracycline (T), Trimethoprim
(TR), Vancomycin (VA).
S
S
R
R
R
R
R
R
R
R
APPENDIX
Appendix ( 4 ) The color of 43 isolates on different media
Isolates code
AE 1
AE 2
AE 3
AE 4
AE 5
AE 6
AE 7
AE 8
AE 9
AE 10
AE 11
AE 12
AE 13
AE 14
AE 15
AE 16
AE 17
AE 18
AE19
AE 20
AE 21
AE22
AE23
AE24
AE25
AE26
AE27
AE28
AE29
AE30
AE31
AE32
AE33
AE34
AE35
AE36
AE37
AE38
AE39
AE40
AE41
AE42
AE 43
O: Orange
Milk
BHIA
NA
Y
W
W
O
Y
W
Y
W
W
Y
W
W
Y
Y
Y
W
W
W
W
W
W
W
W
W
Y
Y
W
W
W
W
W
W
W
W
W
W
W
W
W
Y
W
W
O
W
Y
Y
W
W
W
W
W
W
W
W
O
Y
W
W
W
W
O
Y
W
O
Y
W
O
Y
W
O
Y
W
O
Y
Y
O
Y
Y
Y
Y
Y
O
Y
Y
W
Y
W
O
Y
Y
O
Y
Y
O
Y
Y
O
Y
Y
O
Y
Y
O
Y
Y
O
Y
Y
Y
W
W
W
W
W
O
Y
Y
Y
W
W
Y
W
W
O
Y
Y
Y
W
W
Y: Yellow W: White
MHA
W
Y
W
W
Y
W
W
W
Y
W
W
W
W
W
W
W
W
W
Y
W
Y
Y
Y
Y
Y
Y
Y
Y
W
Y
Y
Y
Y
Y
Y
Y
W
W
Y
W
W
Y
W
Name of Media
CEM
TYA
Y
W
Y
Y
W
Y
W
Y
Y
Y
W
W
W
W
W
W
Y
Y
W
W
W
W
W
W
W
W
W
Y
W
O
W
Y
W
W
W
W
Y
O
W
W
Y
O
Y
O
Y
Y
Y
O
Y
O
Y
O
Y
W
Y
Y
W
W
Y
O
Y
O
Y
O
Y
O
Y
O
Y
O
Y
O
W
Y
W
W
Y
O
W
Y
W
Y
Y
O
W
W
TSA
Y
O
Y
Y
W
W
W
W
Y
W
W
W
W
Y
O
Y
W
W
O
W
O
Y
O
Y
O
O
Y
Y
W
O
O
O
O
O
O
O
Y
W
O
Y
Y
O
Y
PNSA
Y
Y
Y
Y
Y
W
W
W
Y
W
W
W
W
Y
Y
W
W
W
Y
W
O
O
O
O
O
Y
Y
Y
W
O
O
O
O
Y
O
O
Y
W
O
Y
Y
O
Y
SSA
Y
O
Y
W
Y
W
W
W
Y
W
W
W
W
Y
O
Y
W
W
O
W
O
O
Y
W
O
O
Y
Y
W
O
O
Y
O
O
W
O
Y
W
O
Y
Y
Y
Y
Milk: Milk agar medium , BHIA: Brain-Heart Infusion Agar, N.A: Nutrient Agar, MHA: MuellerHinton Agar , CEM: Carotinoid expression medium, TYA:Tripticase yeast medium , TSA:Tripticase
soya medium, PNSA: Peanut seeds medium , SSA:Sesame seed medium , SFSA: Sunflower seeds
medium , ,
SFSA
W
Y
W
W
Y
W
W
W
Y
W
W
W
W
W
Y
Y
W
W
Y
W
Y
Y
Y
Y
Y
Y
Y
Y
W
Y
Y
Y
Y
Y
Y
Y
W
W
Y
W
W
Y
W
‫ﻭﺯﺍﺭﺓ ﺍﻟﺘﻌﻠﻴﻢ ﺍﻟﻌﺎﻟﻲ ﻭﺍﻟﺒﺤﺚ ﺍﻟﻌﻠﻤﻲ‬
‫ﺟﺎﻣﻌﺔ ﺑﻐﺪﺍﺩ ‪ -‬ﻛﻠﻴﺔ ﺍﻟﻌﻠﻮﻡ‬
‫ﻗﺴﻢ ﺍﻟﺘﻘﻨﻴﺎﺕ ﺃﻹﺣﻴﺎﺋﻴﺔ‬
‫ﺩﺭﺍﺳﺔ ﻛﻴﻤﻮﺣﻴﻮﻳﺔ ﻭ ﺟﺰﻳﺌﻴﺔ ﻟﺼﺒﻐﺔ ﺍﻟﺴﺘﺎﻓﻴﻠﻮﺯﺍﻧﺜﻴﻦ ﺍﻟﻤﺴﺘﺨﻠﺼﺔ‬
‫ﻣﻦ ﺑﻜﺘﺮﻳﺎ ﺍﻟﻌﻨﻘﻮﺩﻳﺎﺕ ﺍﻟﺬﻫﺒﻴﺔ ﺍﻟﻤﻌﺰﻭﻟﺔ ﻣﻦ ﻣﺼﺎﺩﺭ ﺳﺮﻳﺮﻳﺔ‬
‫ﺭﺳﺎﻟﺔ‬
‫ﻣﻘﺪﻣﺔ ﺇﻟﻰ ﻛﻠﻴﺔ ﺍﻟﻌﻠﻮﻡ – ﺟﺎﻣﻌﺔ ﺑﻐﺪﺍﺩ‬
‫ﻭﻫﻲ ﺟﺰء ﻣﻦ ﻣﺘﻄﻠﺒﺎﺕ ﻧﻴﻞ ﺩﺭﺟﺔ ﺍﻟﻤﺎﺟﺴﺘﻴﺮ‬
‫ﻓﻲ ﺍﻟﺘﻘﻨﻴﺎﺕ ﺍﻹﺣﻴﺎﺋﻴﺔ‬
‫ﺗﻘﺪﻣﺖ ﺑﻬﺎ‬
‫ﺍﻳﻤﺎﻥ ﺟﻬﺎﺩ ﻧﺎﻓﻊ ﺍﻟﻘﺰﺍﺯ‬
‫ﺑﻜﺎﻟﻮﺭﻳﻮﺱ ﺗﻘﻨﻴﺎﺕ ﺇﺣﻴﺎﺋﻴﺔ ‪ /‬ﻛﻠﻴﺔ ﺍﻟﻌﻠﻮﻡ‬
‫ﺟﺎﻣﻌﺔ ﺑﻐﺪﺍﺩ )‪( ۲۰۰٦‬‬
‫ﺑﺈﺷﺮﺍﻑ‬
‫ﺃ‪.‬ﺩ‪.‬ﺃﻟﻴﺲ ﻛﺮﻳﻜﻮﺭ ﺃﻏﻮﺏ‬
‫ﺃ‪.‬ﻡ‪ .‬ﺩ‪ .‬ﻧﻬﻰ ﺟﻮﺯﻳﻒ ﻗﻨﺪﻻ‬
‫‪۱٤۳٥‬ﻫ‬
‫‪ ۲۰۱٤‬ﻡ‬
‫ﺭﺑﻴﻊ ﺍﻷﻭﻝ‬
‫ﻛﺎﻧﻮﻥ ﺍﻟﺜﺎﻧﻲ‬
‫ﺗﻢ ﻋﺰﻝ ﻭ ﺗﺸﺨﻴﺺ ‪ ٤۳‬ﻋﺰﻟﻪ ﺑﻜﺘﻴﺮﻳﺔ )‪ (%۲۰.۷‬ﺗﻌﻮﺩ ﺍﻟﻰ ﻧﻮﻉ ‪Staphylococcus‬‬
‫‪ aureus‬ﻣﻦ ‪ ۲۰۷‬ﻋﻴﻨﻪ ﺷﻤﻠﺖ ﻋﻴﻨﺎﺕ ﻣﻦ ﺍﻟﺪﻡ ‪ ،‬ﺍﻷﻧﻒ ‪ ،‬ﺍﻟﺠﺮﻭﺡ‪ ،‬ﺍﻹﺩﺭﺍﺭ‪ ،‬ﺍﻟﻤﻬﺒﻞ‪ ،‬ﺍﻻﺫﻥ ﻭ‬
‫ﺍﻟﻌﻴﻮﻥ ﻭ ﺑﻨﺴﺐ ﺗﻮﺍﺟﺪ ﻣﺨﺘﻠﻔﺔ ﺇﺫ ﻛﺎﻧﺖ‬
‫‪(2.33, 6.96, 15.15, 13.95, 16.28, 18.60,‬‬
‫)‪ % 30.23‬ﻋﻠﻰ ﺍﻟﺘﻮﺍﻟﻲ‪ .‬ﻭ ﺍﻅﻬﺮﺕ ﺍﻟﻨﺘﺎﺋﺞ ﺍﻥ ﻋﻴﻨﺎﺕ ﺍﻟﺪﻡ ﻛﺎﻧﺖ ﺍﻻﻛﺜﺮ ﺗﻠﻮﺛﺎ ﺑﻬﺬﺓ ﺍﻟﺒﻜﺘﺮﻳﺎ‬
‫ﺃﻅﻬﺮﺕ ﻧﺘﺎﺋﺞ ﺍﺧﺘﺒﺎﺭ ﺍﻟﺤﺴﺎﺳﻴﺔ ﻟﺨﻤﺴﺔ ﻭ ﻋﺸﺮﻭﻥ ﻣﻀﺎﺩ ﺣﻴﻮﻱ ﻛﻮﻥ ﺟﻤﻴﻊ ﺍﻟﻌﺰﻻﺕ ﻣﻘﺎﻭﻣﺔ‬
‫ﻟﻠﻤﻀﺎﺩﺍﺕ ‪Carbenicillin,‬‬
‫‪Aztreonam,‬‬
‫‪acid,‬‬
‫‪(Amoxicillin/clavulanic‬‬
‫‪Ceftaxime, Cefotaxin, Cifixime, Nitrofurantoin, Pencillin G, Pipracillin,‬‬
‫)‪ Vancomycin‬ﻭ ﺑﻨﺴﺒﺔ ‪% ۱۰۰‬ﻓﻲ ﺣﻴﻦ ﺇﻥ ﻣﻌﻈﻤﻬﺎ ﺃﻅﻬﺮﺕ ﻣﻘﺎﻭﻣﺔ ﻣﺘﻐﺎﻳﺮﺓ ﻟﻠﻤﻀﺎﺩﺍﺕ‬
‫ﺍﻻﺧﺮﻯ ‪ .‬ﺑﻴﻨﻤﺎ ﺍﻅﻬﺮﺕ ﺍﻟﻤﻀﺎﺩﺍﺕ ‪ Imipenem, Trimethoprim‬ﺣﺴﺎﺳﻴﺔ ﺟﻴﺪﻩ ﺍﺗﺠﺎﻩ ﺍﻟﺒﻜﺘﺮﻳﺎ‬
‫ﻣﻤﺎ ﻳﺠﻌﻠﻬﺎ ﺍﻟﻌﻼﺝ ﺍﻻﻣﺜﻞ ﻓﻲ ﺍﻟﻮﻗﺖ ﺍﻟﺤﺎﻟﻲ ‪.‬‬
‫ﻛﺎﻧﺖ ﻧﺴﺒﺔ ﺍﻧﺘﺎﺝ ﺻﺒﻐﻪ ﺍﻟﺴﺘﺎﻓﻴﻠﻮﺯﺍﻧﺜﻴﻦ ﻣﻦ ﻋﺰﻻﺕ ‪ S. aureus‬ﺑﻌﺪ ﺍﺧﺘﺒﺎﺭﻫﺎ ﻋﻠﻰ ﺍﻭﺳﺎﻁ‬
‫ﻣﺨﺘﻠﻔﺔ ﺷﻤﻠﺖ ‪(Milk agar medium ,Trypticase yeast medium , Trypticase‬‬
‫)‪ soya medium ,Peanut seed medium and Sesame seed medium‬ﻛﺎﻻﺗﻲ‬
‫‪ 30.2%، 30.2%، 37.2%، 37.2%، 44.1%‬ﻋﻠﻰ ﺍﻟﺘﻮﺍﻟﻲ‪.‬‬
‫ﺃﺟﺮﻳﺖ ﻋﻤﻠﻴﺔ ﺍﺳﺘﺨﻼﺹ ﺍﻟﺼﺒﻐﺔ ﺑﺎﺳﺘﺨﺪﺍﻡ ﺍﻟﻤﻴﺜﺎﻧﻮﻝ ﻭ ﺗﻤﺖ ﺗﻨﻘﻴﺘﻬﺎ ﺟﺰﺋﻴﺎ ﺑﺎﺳﺘﺨﺪﺍﻡ ﺍﻟﻤﺬﻳﺒﺎﺕ‬
‫ﺍﻟﻌﻀﻮﻳﺔ ﻭ ﺗﻘﻨﻴﺔ ﻛﺮﻭﻣﻮﺗﻮﻏﺮﺍﻓﻴﺎ ﺍﻟﻄﺒﻘﺔ ﺍﻟﺮﻗﻴﻘﺔ ﻭﻭﺟﺪ ﺇﻥ ﻟﻬﺎ ﺛﻼﺙ ﻗﻤﻢ ﻟﻼﻣﺘﺼﺎﺹ ﻭ ﺃﻋﻠﻰ ﻗﻤﺔ‬
‫ﺍﻣﺘﺼﺎﺹ ﻛﺎﻧﺖ ﻋﻨﺪ ﺍﻟﻄﻮﻝ ﺍﻟﻤﻮﺟﻲ‪ ٤٥۰‬ﻧﺎﻧﻮﻣﻴﺘﺮ ‪.‬‬
‫ﺗﻢ ﺍﻧﺘﺨﺎﺏ ﺍﻟﻌﺰﻟﻪ ﺍﻟﻤﺤﻠﻴﻪ ‪ Staphylococcus aureus AE36‬ﻓﻲ ﻫﺬﻩ ﺍﻟﺪﺭﺍﺳﺔ ﻻﻧﺘﺎﺟﻴﺘﻬﺎ‬
‫ﺍﻟﻌﺎﻟﻴﺔ ﻟﺼﺒﻐﺔ ﺍﻟﺴﺘﺎﻓﻴﻠﻮﺯﺍﻧﺜﻴﻦ ﻭﺣﺪﺩﺕ ﺍﻟﻈﺮﻭﻑ ﺍﻟﻤﺜﻠﻰ ﻹﻧﺘﺎﺝ ﺍﻟﺼﺒﻐﺔ ﻣﻦ ﺍﻟﻌﺰﻟﺔ ﺍﻟﻤﺤﻠﻴﺔ ﺍﻟﻤﻨﺘﺨﺒﺔ‬
‫‪ S. aureus AE36‬ﻭﺍﻅﻬﺮﺕ ﺍﻟﻨﺘﺎﺋﺞ ﺃﻥ ﻭﺳﻂ ﺃﻛﺎﺭ ﺍﻟﺤﻠﻴﺐ ﻛﺎﻥ ﺍﻷﻋﻠﻰ ﺇﻧﺘﺎﺟﺎ ﻟﻠﺼﺒﻐﺔ ‪165.21‬‬
‫)ﻭﺣﺪﺓ ‪ /‬ﺧﻠﻴﺔ ( ﻋﻨﺪ ﺍﻟﺮﻗﻢ ﺍﻟﻬﻴﺪﺭﻭﺟﻴﻨﻲ ‪ ، ۸‬ﺑﺤﺠﻢ ‪ ٥۰‬ﻣﻠﻴﻤﺘﺮﺍ ﻣﻦ ﺍﻟﻮﺳﻂ ﺃﻟﺰﺭﻋﻲ ‪،‬ﻋﻨﺪ ﺩﺭﺟﺔ‬
‫ﺣﺮﺍﺭﺓ ‪ ۳۷‬ﺳﻴﻠﺰﻳﻪ ﻭ ﻟﻤﺪﺓ ‪ ۷۲‬ﺳﺎﻋﺔ ‪.‬‬
‫ﺍﻅﻬﺮﺕ ﺍﻟﻨﺘﺎﺋﺞ ﻋﺪﻡ ﻭﺟﻮﺩ ﺍﻱ ﻓﻌﺎﻟﻴﻪ ﻣﻀﺎﺩﺓ ﻟﺼﺒﻐﺔ ﺍﻟﺴﺘﺎﻓﻴﻠﻮﺯﺍﻧﺜﻴﻦ ﻻﻱ ﻣﻦ ﺍﻟﺒﻜﺘﻴﺮﻳﺎ ﺍﻟﻤﺴﺘﺨﺪﻣﻪ‬
‫ﻓﻲ ﻫﺬﻩ ﺍﻟﺪﺭﺍﺳﺔ ‪(Staphylococcus epidermidis, Psedomonas aeruginosa,‬‬
‫‪Salmonella spp., Shigella spp., Esherichia coli, Klebsiella spp and‬‬
‫‪Proteus spp, Pseudomonas fluorescens , Pseudomonas putida ,‬‬
‫)‪Staphylococcus aureus‬‬
‫ﻭﻟﻐﺮﺽ ﺗﺜﺒﻴﻂ ﺍﻟﺘﺎﺛﻴﺮ ﺍﻟﺘﺎﻛﺴﺪﻱ ﻟﻠﺼﺒﻐﺔ ﺍﺗﺠﺎﺓ ﺍﻟﺨﻼﻳﺎ ﺍﻟﻌﺪﻟﻪ )‪ (neutrophil‬ﻓﻲ ﺍﻻﻧﺴﺎﻥ ﻭ‬
‫ﺗﺜﺒﻴﻂ ﺗﺎﺛﻴﺮﻫﺎ ﻛﻌﺎﻣﻞ ﻓﻮﻋﺔ ‪ ،‬ﻁﻔﺮﺕ ﺍﻟﻌﺰﻟﺔ ﺍﻟﻤﺤﻠﻴﺔ ‪ AE36‬ﺍﻟﻤﻨﺘﺠﺔ ﻟﻠﺼﺒﻐﺔ ﺑﺎﺳﺘﺨﺪﺍﻡ ﺍﺛﻴﻞ ﻣﻴﺜﺎﻥ‬
‫ﺳﻠﻔﻮﻧﻴﺖ ﻭﻋﻨﺪ ﻣﻘﺎﺭﻧﺘﻬﺎ ﻣﻊ ﺍﻟﻌﺰﻟﺔ ﺍﻟﻤﺤﻠﻴﺔ ﺍﻟﻐﻴﺮ ﻣﻨﺘﺠﺔ ﻟﻬﺎ ‪ S. aureus AE38‬ﻭ ﻣﻊ ﺍﻟﻌﺰﻟﺔ‬
‫ﺍﻟﻤﺤﻠﻴﺔ ﺍﻻﺻﻠﻴﻪ ‪، S. aureus AE36‬ﺍﻅﻬﺮﺕ ﺍﻟﻨﺘﺎﺋﺞ ﺍﻥ ﺍﻟﺒﻜﺘﺮﻳﺎ ﺗﺼﺒﺢ ﺍﻗﻞ ﺍﻣﺮﺍﺿﻴﺔ ﻋﻨﺪ ﺗﻠﻮﻳﺚ‬
‫ﺟﺮﻭﺡ ﺍﻟﻔﺌﺮﺍﻥ ﺑﻬﺎ ﺑﻌﺪ ﺗﻌﺮﺿﻬﺎ ﻟﻠﻤﻄﻔﺮﺍﺕ ﻭ ﺍﻗﻞ ﺗﺎﺛﻴﺮ ﻋﻠﻰ ﺍﻟﺨﻼﻳﺎ ﺍﻟﻤﻨﺎﻋﻴﺔ ﺍﻟﻌﺪﻟﺔ ﻣﻘﺎﺭﻧﺔ ﺑﺎﻟﻌﺰﻟﻪ‬
‫ﺍﻻﺻﻠﻴﺔ ‪.‬‬
‫ﻳﻤﻜﻦ ﺍﻋﺘﻤﺎﺩ ﺗﻘﻨﻴﺔ ﺗﻔﺎﻋﻞ ﺍﻟﺒﻠﻤﺮﺓ ﺍﻟﻤﺘﺴﻠﺴﻞ ﺍﻟﻠﺤﻈﻲ ﺍﻟﻜﻤﻲ ﻁﺮﻳﻘﺔ ﺩﻗﻴﻘﺔ ﻭﻣﺘﺨﺼﺼﺔ ﻭﺍﻛﺜﺮ‬
‫ﺣﺴﺎﺳﻴﺔ ﻓﻲ ﺍﻟﺘﺤﺮﻱ ﻋﻦ ﺍﻟﻤﻮﺭﺙ ﺍﻟﻤﺸﻔﺮ ﻻﺍﻧﺘﺎﺝ ﺻﺒﻐﺔ ﺍﻟﺴﺘﺎﻓﻴﻠﻮﺯﺍﻧﺜﻴﻦ ﻭ ﻋﻦ ﺍﻟﻤﻮﺭﺙ ﺍﻟﻤﺸﻔﺮ‬
‫ﻟﻤﻘﺎﻭﻣﺔ ﺍﻟﻤﺜﻴﺴﻴﻠﻴﻦ ﺑﺎﺳﺘﺨﺪﺍﻡ ‪. SYBR Green I Real-Time PCR assay‬‬
‫ﺑـــﺎﻗﺔ ﻭﺭﺩ‬
‫ﺍﻟﻬﻲ ﻻ ﻳﻄﻴﺐ ﺍﻟﻠﻴﻞ ﺇﻻ ﺑﺸﻜﺮﻙ ﻭﻻ ﻳﻄﻴﺐ ﺍﻟﻨﻬﺎﺭ ﺇﻻ ﺑﻄﺎﻋﺘﻚ ﻭ ﻻ ﺗﻄﻴﺐ ﺍﻟﻠﺤﻈﺎﺕ ﺇﻻ ﺑﺬﻛﺮﻙ ﻭ ﻻ‬
‫ﺗﻄﻴﺐ ﺍﻵﺧﺮﺓ ﺇﻻ ﺑﻌﻔﻮﻙ ﻭ ﻻ ﺗﻄﻴﺐ ﺍﻟﺠﻨﺔ ﺇﻻ ﺑﻐﻔﺮﺍﻧﻚ‪ ..............‬ﷲ ﺟﻞ ﺟﻼﻟﺔ‪.‬‬
‫ﺇﻟﻰ ﻣﻦ ﺍﺣﻤﻞ ﺍﺳﻤﻪ ﺑﻜﻞ ﻓﺨﺮ‬
‫ﺇﻟﻰ ﻣﻦ ﺍﻓﺘﻘﺪﺗﻪ ﻣﻨﺬ ﺍﻟﺼﻐﺮ‬
‫ﺇﻟﻰ ﻣﻦ ﻳﺮﺗﻌﺶ ﻗﻠﺒﻲ ﻟﺬﻛﺮﻩ ‪ .....‬ﻭﺍﻟﺪﻱ )ﺭﺣﻤﻪ ﷲ (‬
‫ﺇﻟﻰ ﻣﻦ ﺭﺁﻧﻲ ﻗﻠﺒﻬﺎ ﻗﺒﻞ ﻋﻴﻨﻴﻬﺎ‬
‫ﻭ ﺣﻀﻨﺘﻨﻲ ﺃﺣﺸﺎﺅﻫﺎ ﻗﺒﻞ ﻳﺪﻳﻬﺎ‬
‫ﻭﺍﺧﺺ ﷲ ﺍﻟﺠﻨﺔ ﺗﺤﺖ ﻗﺪﻣﻴﻬﺎ ‪ .....‬ﺃﻣﻲ )ﺣﻔﻈﻬﺎ ﷲ (‬
‫ﺇﻟﻰ ﺗﻮﺃﻡ ﺭﻭﺣﻲ ﻭ ﺭﻓﻴﻖ ﺩﺭﺑﻲ ﺇﻟﻰ ﺻﺎﺣﺐ ﺍﻟﻘﻠﺐ ﺍﻟﻄﻴﺐ‬
‫ﺇﻟﻰ ﺭﻣﺰ ﺍﻟﻮﻓﺎء ﻭﺍﻹﺧﻼﺹ ‪ .....‬ﺯﻭﺟﻲ ﺍﻟﻌﺰﻳﺰ‬
‫ﺇﻟﻰ ﻣﻦ ﺃﺭﻯ ﺍﻟﺘﻔﺎﺅﻝ ﻓﻲ ﻋﻴﻨﻬﻢ ﻭ ﺍﻟﺴﻌﺎﺩﺓ ﻓﻲ ﺿﺤﻜﺘﻬﻢ‬
‫ﺇﻟﻰ ﺍﻟﻮﺟﻮﻩ ﺍﻟﻤﻔﻌﻤﺔ ﺑﺎﻟﺒﺮﺍءﺓ ‪ .....‬ﺃﻁﻔﺎﻟﻲ‬
‫) ﺟﻨﺔ ﻭ ﺷﻬﺪ(‬
‫ﺇﻟﻰ ﻣﻦ ﺑﻬﻢ ﺍﻛﺒﺮ ﻭ ﻋﻠﻴﻬﻢ ﺍﻋﺘﻤﺪ ﺑﻌﺪ ﷲ‬
‫ﺇﻟﻰ ﺍﻟﺸﻤﻮﻉ ﺍﻟﺘﻲ ﺗﻨﻴﺮ ﻅﻠﻤﺔ ﺣﻴﺎﺗﻲ ‪ .....‬ﺃﺧﻮﺍﺗﻲ ﻭ ﺇﺧﻮﺗﻲ‬
‫)ﺭﻧﺎ ‪ ،‬ﻓﺎﺗﻦ ‪ ،‬ﺇﺑﺮﺍﻫﻴﻢ ‪،‬ﺍﺣﻤﺪ(‬
‫ﺇﻳﻤﺎﻥ ﺟﻬﺎﺩ ﺍﻟﻘﺰﺍﺯ‬