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Micrococcaceae Chapter 14 Staphylococci Family Micrococcaceae • Includes 4 genera: – Planococcus – free living saprophyte – Micrococcus – free living saprophyte – Stomatococcus – normal flora on surface of primates and other mammals – Staphylococcus – normal flora on surface of primates and other mammals • All, except Planococcus, isolated from clinically significant sources Infection and Disease • Stomatococci – oral normal flora (NF), emerging pathogen in immunocompromised • Micrococci - pathogen when accidentally introduced into susceptible host • Staphylococci - long recognized as important human pathogen, most commonly isolated species in order of pathogenicity: – S. aureus – S. epidermidis – S. saprophyticus. Gram Stain • Gram (+) coccus • Lose ability to retain Gram (+) staining characteristic with longer culture • Occur singly, pairs, tetrads (common for Micrococci), or clusters • Staphylococci – staphyle (bunch of grapes), arrangement due to cell division in different planes Laboratory Culture • Grow on most lab media that support growth of Gram (+) microorganisms (MO) • Overnight culture: smooth, circular, buttery colonies • S. aureus - golden or yellow, many clinical isolates creamy • S. epidermidis - white • M. luteus - bright yellow Differential Media: Columbia Blood Agar (CBA) • S. aureus produces complete (beta) hemolysis • Other Staphylococcus species produce partial (alpha) hemolysis or no hemolysis (gamma) Selective Media: PEA, CNA • Used to isolate Staph. from specimens heavily contaminated • Phenyl Ethyl Alcohol (PEA) – inhibit Gram (-) bacteria • Columbia CNA Agar antibiotics Colistin and Nalidixic Acid to inhibit Gram (-) bacteria Differential & Selective Media: Mannitol Salt Agar (MSA) • High salt (7.5%) inhibit growth most MO • Staph. are halophiles and grow in up to 10% NaCl • Also contains mannitol and pH indicator phenol red (pH 7 = red) • If MO growing on MSA ferments mannitol, the acid produced turns phenol red = yellow • S. aureus ferments mannitol • S. epidermidis does not Biochemical Identification: Catalase Test • Stomatococci = catalase (-) • Micrococci and Staphylococci = catalase (+) Oxidative/Fermentative (O/F ) Media • Test done using two tubes of O/F: – Oxidation - open to air; (+) = yellow – Fermentation – overlay with mineral oil, absence of O2; (+) = yellow • Micrococci = oxidative or inert (asaccharolytic) • Staphylococci = oxidative and fermentative Biochemical Test: Micrococci vs Staphylococci • Modified Oxidase Test (contains DMSO allows penetration of test reagent through thick G+ cell wall): – Micrococci = (+) – Staphylococci = (–) • Lysostaphin (protease breaks glycine peptide linkage in cell: – Micrococci = Resistant – Staphylococci = Sensitive • Bacitracin – (0.04 units, • Taxo A disc): – Micrococci = Sensitive – Staphylococci = Resistant ID Staphylococcus aureus: Coagulase Test • In presence of coagulase, fibrinogen converted to fibrin • Staphylococcus aureus = coagulase (+) • Other species of Staph. are coagulase positive, but are rare isolates from human infections (i. e., S. intermedius from canine bites) • Therefore, (+) coagulase test usually identifes isolate as S. aureus • All other Staph species collectively called coagulase negative Staph. (CoNS), these include S. epidermidis and S. saprophyticus Two Coagulase Test • Slide test for bound coagulase • Tube test test for free or extracellular coagulase Differentiation of Staphylococcus • Mannitol fermentation – S. aureus and some S. saprophyticus = (+) – S. epidermidis = (-) • DNAse – S. aureus = (+) – S. epidermidis and S. saprophyticus = (-) Differentiation of Coagulase Negative Staphylococcus: Novobiocin • Usually for urinary tract isolates • ID S. epidermidis from S. saprophyticus • Novobiocin (5 ug disk) • S. epidermidis = Sensitive, zone of inhibition ≥17 mm. in diameter • S. saprophyticus = Resistant, zone <17 mm. in diameter Serology & Phage Test • Antibody titer to Staph. teichoic acids (part of the cell wall); rising titer may be used to confirm diagnosis of Staph. endocarditis • Epidemiologic investigations identify different strains of S. aureus based on type of antigenic polysaccharide capsule • Phage typing – S. aureus and S. epidermidis strains placed into different groups based on susceptibility by different bacteriophages; used in epidemiologic investigations Mechanisms of Pathogenicity: Staphylococcus aureus • Capsule – allows MO to resist phagocytosis; some only form capsules “in vivo” • Teichoic acids –a fibronectin binding protein; involved in adherence, as fibronectin on surface of many cells • Protein A – binds to Fc region of IgG; inhibits phagocytosis by preventing opsonization (antibody-enhanced phagocytosis) S. aureus Exotoxins • • • • • Hemolysin Panton-Valentine Leukocidan Enterotoxin Exfoliative Toxic Shock Syndrome Toxin (TSST) Exotoxins • Hemolysin – S. aureus is beta hemolytic: – May produce 4 different hemolysins: ,, , – Except for hemolysin, they lyse RBCs and also leukocytes and tissue cells – toxin, in particular, produce extensive tissue damage – hemolysin known as hot-cold lysin; hemolytic activity enhanced when 370 C followed by 40 C incubation • Panton-Valentine Leukocidan: – Acts exclusively on WBCs (PMNs and macrophages) – Lytic activity due to alteration of Na+ - K+ pump, leads to altered permeability and eventual lysis of cell Exotoxins • Enterotoxins – 8 distinct enterotoxins responsible for Staph. food poisoning: – Heat stable – Stimulate neural receptors in G.I. tract causing pain, vomiting, diarrhea within 6 hours of ingestion – Symptoms short lived – Former type F (see below) now known as toxic shock syndrome toxin (TSST-1) • Exfoliative (Epidermolytic) toxin: – Cleaves upper layer of the epidermis – Condition called scalded skin syndrome (SSS) • TSST– 1: – Pyrogenic (fever causing) due to IL-1 induction – Erythroderma (red skin) – Enhanced susceptibility to endotoxin shock • Many of the effects of enterotoxin, exfoliative toxin and TSST-1 are due to their action as a superantigen Action of a Superantigen • Non-specific stimulation of T cells • Leading to cytokine release (“cytokine storm”) • Causes inflammation, leads to fever, hypotension and shock S. aureus Enzymes • • • • • • • Coagulase Staphylokinase Hyaluronidase Lipase Beta lactamase DNAse Slime production Enzymes: Coagulase • Helps to wall off MO from host immune system • Also help by coating neutrophils with fibrin to protect MO from phagocytosis • Many Staph. infections are characterized by abscess formation • Free or extracellular coagulase combines with serum component to produce thrombin-like activity to cleave fibrinogen to form fibrin clot • Bound coagulase binds to fibrinogen on cell surfaces converting it to fibrin, producing fibrin clots and causing agglutination of the bacterial cells (also called clumping factor) • High concentrations of coagulase can lead to intravascular coagulation, particularly in the lungs Enzymes • Staphylokinase – dissolves fibrin clots that host may lay down during an inflammatory reaction to try to wall off the infection; help MO to disseminate • Hyaluronidase – depolymerizes hyaluronic acid, the ground substance of tissues; help MO to disseminate • Lipase – hydrolyzes lipids; help MO to disseminate • Beta lactamase (Cefinase disks) – breaks beta lactam ring to inactivate penicillin Enzymes • DNAse – degrades accumulated inflammatory exudate DNA from leukocyte disintegration, helping MO to spread (DNA very viscous making dissemination difficult) • Slime production – extracellular glycoconjugate helps MO to adhere to smooth surfaces and produced by CNS; important in colonization of indwelling catheters Summary: S. aureus Virulence Factors Clinical Significance of Staphylococcus • Ubiquitous, found as NF of humans and other animals • CoNS strains (usually S. epidermidis) NF of skin • S. aureus NF of nasopharynx in 10-40% of population; percentage higher in hospitalized patients • Opportunistic (S. epidermidis) or Facultative (S. aureus) pathogens S. aureus : Invasive Infections • Cause localized infections in nearly any area of body • Local skin infections most common • Suppuration (pus production) hallmark of these infections • Folliculitis - infection of hair follicle; if hair follicle is eyelash, infection called a stye • Furuncle or boil – folliculitis spreads to involve subcutaneous tissue • Carbuncle – series of interconnected furuncles • • • • • • • Invasive Infections Spreads throughout body Bacteremia Septicemia with lymphangitis Osteomyelitis Pneumonia Meningitis Endocarditis – acute or subacute (can occur following tooth extraction) • S. aureus and S. saprophyticus cause urinary tract infection • S. saprophyticus is second most common cause of urinary tract infection in sexually active young women S. aureus :Toxigenic Diseases • Food poisoning due to heat stable enterotoxin; more common in foods with mayonnaise or custard • Toxic shock syndrome – commonly starts as vaginal infection in menstruating women using absorbent tampons; sudden onset of high fever, vomiting, diarrhea, red rash, shock due to TSST-1; followed 1-2 weeks by desquamation of palms of hands and soles of feet • Scalded skin syndrome: – Exfoliative toxin, initially causes red rash, followed by peeling away of the skin in sheaths – Usually occurs 2 times, but heals without scarring – More common in infants and young children Summary: Staphylococcus Infections Antimicrobial Treatment • Most S. aureus (85%) now produce beta lactamase to inactivate penicillin • Methicillin, a beta lactamase resistant penicillin used • Now methicillin resistant strains of S. aureus (MRSA): – change in cell wall and penicillin binding proteins (enzymes for peptidoglycan cross-linking) – altered binding of antibiotics, no longer inhibit enzymes – also resistance to cephalosporins, streptomycin, tetracycline, sulfonamides • For MRSA use of vancomycin: – given I.V.,requires hospitalization – Vancomycin resistant strains have now been reported! • Other Staphylococcus species tend to be even more resistant to antibiotics than S. aureus, so antimicrobial sensitivity testing essential Experimental Treatment: Bacterial Interference • For individuals with chronic infections of S. aureus • The individual is colonized with a S. aureus strain of low virulence • Idea that no superinfection will occur if the individual is already colonized Class Assignment • Textbook Reading: Chapter 14 Staphylococci • Key Terms • Learning Assessment Questions Case Study 1 Staphylococcus • An 18-year-old man fell on his knee while playing basketball. The knee was painful, but the overlying skin was unbroken. • The knee was swollen and remained painful the next day, so he was taken to the local emergency department. • Clear fluid was aspirated from the knee, and the physician prescribed symptomatic treatment. Case Study 1 • Two days later, the swelling returned, the pain increased, and erythema developed over the knee. • Because the patient also felt systemically ill and had an oral temperature of 38.8°C, he returned to the emergency department. • Aspiration of the knee yielded cloudy fluid, and cultures of the fluid and blood were positive for S. aureus. Case Study 1 - Questions • 1. Name two possible sources of this organism. • 2. Staphylococci cause a variety of diseases, including cutaneous infections, endocarditis, food poisoning, SSS, and TSS. How do the clinical symptoms of these diseases differ from the infection in this patient? Which of these diseases are intoxications? Case Study 1 - Questions • 3. What toxins have been implicated in staphylococcal diseases? Which staphylococcal enzymes have been proposed as virulence factors? • 4. What is the antibiotic of choice for treating staphylococcal infections? (Give two examples.) • Written Report: – Short reply to each question (2-3 sentences) – Due Jan. 11 in lecture – No late reports or email