Republic of Iraq Ministry of Higher Education And Scientific Research University of Baghdad
Transcription
Republic of Iraq Ministry of Higher Education And Scientific Research University of Baghdad
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. 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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