1. Skin infection by S.aureus-ppt.pptx.pdf

Study of Skin Infections  Introduction Skin is the largest organ of the body The temperature is less than 370 C (Skin surface) Maintain homeostasis eg. pH The skin acts as the first line of defense to protect the entry from microbes. ...

Study of Skin Infections  Introduction Skin is the largest organ of the body The temperature is less than 370 C (Skin surface) Maintain homeostasis eg. pH The skin acts as the first line of defense to protect the entry from microbes.  Layers of the Skin The epidermis(stratum cornium), the outermost layer of skin, provides a waterproof barrier and creates our skin tone. The dead skin cells are present and shedded time to time preventing long time settling of organisms The dermis, the largest layer beneath the epidermis, contains tough connective tissue, hair follicles, sebaceous gland and sweat glands. The deeper subcutaneous tissue (hypodermis) is made of fat and connective tissue.  Mechanism of skin as a barrier for organisms Stratum cornium layer where the dead cells are continuously shedded Sweat glands secrete sweat which has lysozyme (acts on bacterial cell wall) Sebaceous gland secretes sebum that lubricates the skin and acts hydrophobically. Sebum contains sapienic acid that helps to destroy some of the gram positive bacteria.  What helps the organism to gain entry? Skin pores( hair follicles) waxing. Wounds (scratches, cuts, burns) Mechanical damage Insect or animal bites Surgical damage  Normal Microbiota of Skin Microorganisms that find the skin a satisfactory environment are resistant to drying and to relatively high salt concentrations.. The skin’s normal microbiota contain relatively large numbers of gram-positive cocci, such as staphylococci and micrococci. Vigorous washing can reduce their numbers but will not eliminate them. Microorganisms remaining in hair follicles and sweat glands after washing will soon reestablish the normal populations.  Normal Microbiota of Skin Areas of the body with more moisture, such as the armpits. have higher populations of microbes. These metabolize secretions from the sweat glands and are the main contributors to body odor Some diphtheroids, such as Cutibacterium (Propionibacterium) acnes are typically anaerobic and inhabit hair follicles. Their growth is supported by secretions from the oil glands (sebum), which, makes them a factor in acne  Normal Microbiota of Skin A yeast, Malassezia furfur, is capable of growing on oily skin secretions and is associated with the scaling skin condition known as dandruff. Shampoos for treating dandruff contain the antibiotic ketoconazole or zinc pyrithione or selenium sulfide. Some common infections of skin  Staphylococcus aureus Morphology - Staphylococci are spherical cells about 1 μm in diameter arranged in irregular clusters. - Single cocci, pairs, tetrads, and chains. - Gram positive - Staphylococci are nonmotile and do not form spores. - They have capsule.  Cultural characterictics Staphylococci grow readily on most bacteriologic media They grow most rapidly at 37°C but form pigment best at room temperature (20–25°C). S aureus usually forms gray to deep golden yellow colonies. On blood agar S.aureus shows beta hemolysis. Staphylococci slowly ferment many carbohydrates, producing lactic acid but not gas. The staphylococci produce catalase The organism is isolated by streaking material from the clinical specimen (or from a blood culture) onto solid media such as blood agar, tryptic soy agar, or heart infusion agar. Mannitol Salt Agar (MSA), a selective media, S. aureus being a mannitol fermenting bacteria, gives yellow or gold colonies. Cultural characterictics  Pathogenesis S. aureus infects the skin, it stimulates a vigorous inflammatory response, and macrophages and neutrophils are attracted to the site of infection. Most strains secrete a protein that blocks chemotaxis of neutrophils to the infection site, and if the bacterium does encounter phagocytic cells, it often produces toxins that kill them. It is resistant to opsonization , but, failing this, it can survive well within the phagosome. Other proteins it secretes neutralize the antimicrobial peptide defensins on skin, and its cell wall is lysozyme resistant. All humans possess antibodies against S. aureus, but they do not effectively prevent repeated infections.  Virulent Factors -Enzymes and Toxins 1.Coagulase - S. aureus produces an extracellular coagulase, an enzyme like protein that clots oxalated or plasma. - Coagulase binds to prothrombin; together they become enzymatically active and initiate fibrin polymerization. - Coagulase may deposit fibrin on the surface of staphylococci, perhaps altering their ingestion by phagocytic cells or their destruction within such cells.  Virulent Factors -Enzymes and Toxins 1. Coagulase  Virulent Factors -Enzymes and Toxins 2.Catalase - Staphylococci produce catalase, which converts hydrogen peroxide into water and oxygen. - This catalase produced by S.aureus destroys hydrogen peroxide produce by the phagocyte during phagocytosis. - The catalase test differentiates the staphylococci, which are positive, from the streptococci, which are negative.  Virulent Factors -Enzymes and Toxins 3. Hyaluronidase and Collagenase - Collagenase and hyaluronidase degrade collagen and hyaluronic acid, respectively, thereby allowing the bacteria to spread through subcutaneous tissue.  Virulent Factors -Enzymes and Toxins - Action of Collagenase and hyaluronidase.  Virulent Factors -Enzymes and Toxins 3.Other Enzymes - Other enzymes produced by staphylococci include a or spreading factor—a staphylokinase resulting in fibrinolysis but acting much more slowly than streptokinase, proteinases, lipases, and β-lactamase..  Virulent Factors -Enzymes and Toxins 4.Exotoxins - comprises of four toxins α,β,γ,δ: also called hemolysin, – α exotoxins- heterogenous protein acts on a broad spectrum of eukaryotic cell membranes – β exotoxins- degrades sphingomyelin and also toxic for many kinds of cells, including human red blood cells – γ exotoxins- interact with two proteins to form six potential two-component toxins. All six toxins lyse WBC by pore formation in the cellular membranes that increase cation permeability. – δ exotoxins- disrupts biological membrane  Virulent Factors -Enzymes and Toxins 5.Panton–Valentine Leukocidin - composed of two components S and F which act synergistically to kill white blood cells - It can kill white blood cells of humans and rabbits. - PVL is a bacteriophage encoded biocomponent leukotoxin  Virulent Factors -Enzymes and Toxins 6.Exfoliative toxins - Exfoliative toxin is encoded by eta gene - These are epidermolytic toxins(causes separation of epidermis and dermis) -Causes Scalded skin syndrome that occurs in infants - These epidermolytic toxins yield the generalized desquamation of the staphylococcal scalded skin syndrome by dissolving the mucopolysaccharide matrix of the epidermis.  Virulent Factors -Enzymes and Toxins Scalded Skin syndrome - Epidermolytic toxins (causes separation of epidermis and dermis)  Virulent Factors -Enzymes and Toxins 7.Toxic Shock Syndrome Toxin - TSST-1 binds to major histocompatibility class (MHC) class II molecules, yielding T-cell stimulation, which promotes the protean manifestations of the toxic shock syndrome - The toxin is associated with fever, shock, and multisystem involvement, including a desquamative skin rash -The gene for TSST-1 is found in about 20% of S aureus isolates.  Virulent Factors -Enzymes and Toxins 8.Enterotoxins - There are 15 enterotoxins (A–E, G–P) that, similar to TSST-1, are superantigens. - Approximately 50% of S aureus strains can produce one or more of them. - Important causes of food poisoning, enterotoxins are produced when S aureus grows in carbohydrate and protein foods.  Virulent Factors -Genes responsible for antibiotic resistance mecA gene - Provides resistance to Methicillin , Oxacillin and Penicillin and m any other drugs too. vanA gene -Resistance to Vancomycin  Pathogenesis -A localized staphylococcal infection appears as a “pimple,” hair follicle infection, or abscess. -Groups of S aureus established in a hair follicle lead to tissue necrosis -Coagulase is produced and coagulates fibrin around the lesion and within the lymphatics, resulting in formation of a wall that limits the process and is reinforced by the accumulation of inflammatory cells and, later, fibrous tissue. -The wall of fibrin and cells around the core of the abscess tend to prevent spread of the organisms and should not be broken down by manipulation or trauma -Within the center of the lesion, liquefaction of the necrotic tissue occurs (enhanced by delayed hypersensitivity), and the abscess “points” in the direction of least resistance. -Staphylococci of low invasiveness are involved in many skin infections (eg, acne, pyoderma, or impetigo).  Pathogenesis - S aureus infection can also result from direct contamination of a wound, such as a postoperative staphylococcal wound infection or infection after trauma (chronic osteomyelitis subsequent to an open fracture, meningitis after skull fracture).  Clinical manifestation - Abscess, painful Pimple, -Boils, Impetigo, -Cellulitis -Scalded skin syndrome -Folliculitis Infections caused by S.aureus  Laboratory Diagnosis 1.Specimen Collection -Surface swab pus or aspirate from an abscess -Transport medium such as Tryptic soy broth (TSB) can be used 2. Smears -Typical staphylococci appear as gram-positive cocci in clusters in Gram-stained smears of pus. 3. Culturing -Specimens plated on blood agar plates give rise to typical colonies in 24 hours at 37°C shows beta hemolysis due to Hemolysin enzyme. -Salt Mannitol agar can be used to observe typical pigmented colonies of S.aureus after 48 hrs at 37°C  Laboratory Diagnosis 4.Catalase Test -This test is used to detect the presence of -cytochrome oxidase enzymes. -A drop of 3% hydrogen peroxide solution is placed on - a slide,and a small amount of the bacterial growth is placed in the solution. -The formation of bubbles (the release of oxygen) - indicates a positive test result.  Laboratory Diagnosis 5.Coagulase test -Citrated rabbit (or human) plasma diluted 1:5 is mixed with an equal volume of broth culture or growth from colonies on agar and incubated at 37°C. -A tube of plasma mixed with sterile broth is included as a control. If clots form in 1–4 hours, the test result is positive  Laboratory Diagnosis 6.Biochemical tests -S.aureus is able to ferment following sugars -1.Maltose -2.Trehalose -3.Sucrose -4.Mannitol -S.aureus gives Methyl Red test positive which is done for detecting presence of Acid formed during metabolism using mixed acid fermentation pathway. -It also gives VP(Voges Proskauer) test positive -It gives citrate test positive as well.Citrate test is used to determine the ability to use citrate as sole carbon source  Biochemicals Citrate test -The citrate test detects the ability of an organism to use citrate as the sole source of carbon and energy. -Bacteria are inoculated on a medium containing sodium citrate and a pH indicator such as bromothymol blue. -The medium also contains inorganic ammonium salts, which are utilized as sole source of nitrogen. Use of citrate involves the enzyme citrase, which breaks down citrate to oxaloacetate and acetate. -Production of sodium bicarbonate (NaHCO3) as well as ammonia(NH3) from the use of sodium citrate and ammonium salts results in alkaline pH. This results in a change of the medium's color from green to blue. Bacterial colonies are picked up from a straight wire and inoculated into slope of Simmons citrate agar and incubated overnight at 37 °C. If the organism has the ability to use citrate, the medium changes its color from green to blue.  Biochemicals 7) Carbohydrate Fermentations - Fermentation media are used to differentiate organisms based on their ability to ferment carbohydrates incorporated into the basal medium. -Phenol Red Broth Medium with various added carbohydrates serves as a differential medium by aiding in differentiation of various species and genera by their ability to ferment the specific carbohydrate, with the production of acid or acid and gas. - The carbohydrate source can vary based on test requirements. The common broth media used are: Phenol Red Glucose Broth Phenol Red Lactose Broth Phenol Red Maltose Broth Phenol Red Mannitol Broth Phenol Red Sucrose Broth  Biochemicals Carbohydrate Fermentations - The medium may contain various color indicators. In addition to a color indicator to detect the production of acid from fermentation, a Durham tube is placed in each tube to capture gas produced by metabolism. - The carbohydrate fermentation patterns shown by different organisms are useful in differentiating among bacterial groups or species. -Phenol Red Broth is a general-purpose differential test medium typically used to differentiate gram negative enteric bacteria. It contains peptone, phenol red (a pH indicator), a Durham tube, and one carbohydrate (glucose, lactose, or sucrose). -Phenol red is a pH indicator which turns yellow below a pH of 6.8 and fuchsia above a pH of 7.4. If the organism is able to utilize the carbohydrate, an acid by-product is created, which turns the media yellow. -If the organism is unable to utilize the carbohydrate but does use the peptone, the by-product is ammonia, which raises the pH of the media and turns it fuchsia.  Biochemicals 7) Carbohydrate Fermentations Procedure 1) Innoculate each tube with test organism with wireloop. 2) Incubate the tubes at 37C for 24 hrs. 3) Check the color change and gas formation.  Urease test 1. Urea Agar was developed by Christensen in 1946 for the differentiation of enteric bacilli. The urease test is used to determine the ability of an organism to split urea, through the production of the enzyme urease. Principle - Urea is the product of decarboxylation of amino acids. Hydrolysis of urea produces ammonia and CO2. - The formation of ammonia alkalinizes the medium, and the pH shift is detected by the color change of phenol red from light orange at pH 6.8 to magenta (pink) at pH 8.1. - Rapid urease-positive organisms turn the entire medium pink within 24 hours. Urease test  TEST S taphylococcus aureus Glucose (A) A Glucose (An) - Mannitol (A) A Mannitol (A) A Coagulase + Catalase + Urease - Citrate + 7.5% NaCl +  Treatment -Sometime there is requirement of surgical removal of dead tissue in case of Necrosis -Drainage of pus from infection site -Staphylococcus.aureus are becoming increasingly resistant to many commonly used antibiotics. -Penicillin with beta-lactamase-inhibitors are used to treat S.aureus infections such as Amoxicillin + clavulonic acid -Other options are Clindamycin and Rifampicin -Sometime there is requirement of surgical removal of dead tissue in case of Necrosis -Drainage of pus from infection site.  Prevention -Wash hands often ,before and after touching broken skin -Antibiotic ointments Eg. Fusidic acid or Mupirocin may be used which are available as OTC -Maintaining hygienic conditions THANK YOU!