Staphylococcus and Micrococcus Lecture Notes PDF

OUR LADY OF FATIMA UNIVERSITY – ANTIPOLO CAMPUS COLLEGE OF MEDICAL LABORATORY SCIENCE CLINICAL BACTERIOLOGY 211 LECTURE GRAM POSITIVE COCCI Morphologically round in shape Violet/purple GRAM POSITIVE COCCI Kingdom Monera Monera Family Micrococcaceae Streptococcaceae...

OUR LADY OF FATIMA UNIVERSITY – ANTIPOLO CAMPUS COLLEGE OF MEDICAL LABORATORY SCIENCE CLINICAL BACTERIOLOGY 211 LECTURE GRAM POSITIVE COCCI Morphologically round in shape Violet/purple GRAM POSITIVE COCCI Kingdom Monera Monera Family Micrococcaceae Streptococcaceae Genus Streptococcus Micrococcus Enterococcus Staphylococcus Aerococcus Stomatococcus Leuconcoccus Planococcus Gemella Pediococcus  MICROCOCCUS STAPHYLOCOCCUS STREPTOCOCCUS Morphology: Gram Pairs, tetrads, irreg. Clusters Chains Stain clusters Growth characteristics Obligate aerobe Facultative Facultative anaerobe (5% SDA) anaerobe Colonies Small-medium Medium-large Small (pinpoint) (pinhead) Hemolysis Non-hemolytic Beta-hemolysis Alpha, Beta, Gamma Non-hemolytic hemolysis Catalase test + + - O/F test Non-glucose fermenter Glucose fermenter (-) (+) Glucose oxidizer (+) Glucose oxidizer (+) Modifies Oxidase Test + - - Lystostaphin R S V Furazolidone (100 ug R S V disk) Bacitracin (0.04 ug disk) S R Group A = S Others = R Staphylococcus spp. STAPHLE  u Two groups: 1. Coagulase-positive Staphylococci a) S. aureus – golden-yellow colonies; pathogenic 2. Coagulase-negative Staphylococci (CoNS) a) S. aureus – lemon yellow colonies; chromogenic opportunistic pathogen b) S. citreus – white pigment; chromogenic opportunistic pathogen Staphylococcus c) S. albus – white pigment spp. d) S. hyicus – animal pathogen e) S. intermedius – animal pathogen f) S. epidermidis – predominant normal flora on the skin; leading cause of Iantrogenic infection g) S. saprophyticus – opportunistic pathogen; normal flora of skin; frequently cause UTI, abortion/miscarriages h) S. lugdunensis – opportunistic pathogen; normal flora i) S. haemolyticus – recovered in wounds, UTIs  General Characteristics of Staphylococci u Common inhabitant of the skin and mucous membranes, responsible for several suppurative infections u Gram-positive cocci, catalase-positive u Spherical cells arranged in irregular clusters, appear singly, in pairs u Nonmotile, non-spore forming u Aerobic or facultatively anaerobe EXCEPT for S. saccharolyticus (obligate anaerobe) u Lack spores and flagella (non-motile), may have capsules  General characteristics of Staphylococci u Colonies are medium sized (4 to 8 mm) and appear cream-colored, white or rarely light gold, and “buttery-looking” u Some species are β-hemolytic u Rare strains of staphylococci are fastidious requiring carbon dioxide, hemin, or menadione for growth. u so-called small colony variants grow on media containing blood, forming colonies about 1/10 the size of wild-type strains after at least 48 hours of incubation. Staphylococcus aureus u Most clinically significant species in human u Coagulase-positive u It causes various cutaneous infections and purulent abscesses. u These cutaneous infections can progress to deeper abscesses involving other organ systems and produce bacteremia and septicemia.  u Subdivided into two groups based on their novobiocin susceptibility pattern: 1. Novobiocin susceptibility includes Coagulase- S. Epidermidis, S. Capitis, S. Haemolyticus, S. Hominis subsp. negative Hominis, S. Lugdunensis, S. staphylococci Saccharolyticus, S. Warneri, and (CoNS or non- other species staphylococcus 2. Novobiocin resistant group aureus) consists of such species as S. Cohnii, S. Kloosii, S. Saprophyticus, and S. Xylosus Other coagulase-producing Staphylococci u S. intermedius u S. pseudintermedius u S. hyicus u S. delphini u S. lutrae u S. agnetis u Some strains of S. schleiferi u S. lugdunensis and S. schleiferi – mistaken as coagulase positive staphylococci because of the presence of clumping factor  SPHINGOMYELINASE C 1. Cytolytic Toxins a. Hemolysin – (alpha, beta, delta, gamma) Virulence uPathogenic factor; destroys Factors of S. RBC Aureus b. Panton-Valentine Toxin/Leukocidin uRupture of WBCs  2. Enterotoxins – heat stable (100° C for 30 minutes); cause various symptoms Virulence inc. diarrhea and vomiting Factors of S. a. Enterotoxin A, B and D – cause of food poisoning Aureus  3. Toxic Shock Syndrome Toxin-1 (TSTT-1) u Formerly known as Enterotoxin F and pyrogenic exotoxin C u Causes: Toxic Shock Syndrome Virulence u The enterotoxins and TSST-1 belong Factors of S. to a class of polypeptides known as superantigens which are potent Aureus activators of T lymphocytes leading to the release of cytokines such as interleukins and tumor necrosis factor.  4. Epidermolytic Toxin (Exfoliative Toxin) Also known as ET or “exfoliatin” Virulence u Responsible for the staphylococcal Factors of S. u scalded skin syndrome (SSSS) Aureus u It is also has been implicated in bullous impetigo EXFOLIATIVE TOXIN –A EXFOLIATIVE TOXIN –B  5. Fibrolysin/Staphylokinase – responsible for lysin clot 6. Protein A and Polysaccharide A – responsible for anti-phagocytosis 7. Staphylocoagulase – localizes abscess; Virulence virulence factor Factors of S. 8. Hyaluronidase – degrades hyaluronic acid components of tissue; spreading factor Aureus 9. Lipase – spreading factor 10. DNAse – spreading factor u Other enzymes produced which are used to identify S. aureus: Gelatinase, Thermonuclease, Penicillinase, Catalase Infections caused by Staphylococcus aureus A. Cutaneous Infections (Skin and Wound Infections) u Infections caused by S. aureus are suppurative u The abscess is filled with pus and surrounded by necrotic tissues and damaged leukocytes u Common skin infections caused by S. aureus: 1. Folliculitis – inflammation of hair follicle or oil gland; infected area is raised and red 2. Furuncles (Boils) – extension of folliculitis; large, raised and superficial abscess 3. Carbuncles – cluster of boils 4. Bullous impetigo – larger than streptococcal nonbullous impetigo; surrounded by a small zone of erythema  u Include osteomyelitis, periostitis, tonsillitis, pharyngitis, sinusitis, bronchopneumonia, empyema, B. Deep septicemia, meningitis, endocarditis, Infections breast abscess, renal abscess and abscesses in other organs.  1. Food Poisoning u The enterotoxins do not cause any detectable odor or change in the appearance or taste of the food u Symptoms appear rapidly (approximately 2 to 8 hours after ingestion of the food) and resolve within 24 to 48 hours u Nausea, vomiting, abdominal pain and severe C. Toxin- cramping (common) mediated u Diarrhea and headaches (rare) No fever Diseases u 2. Toxic Shock Syndrome (TSS) u Fatal, multisystem disease characterized by sudden onset of fever, chills, vomiting, diarrhea, muscle aches and rash (predominantly on the trunk) u Progress to hypotension and shock u Most cases occur in menstruating women who use tampons  C. Toxin-mediated Diseases 3. Exfoliative Diseases u Lesions are produced by the strains of S. aureus which produce epidermolytic toxins u Responsible for the ‘staphylococcal scalded skin syndrome’ (SSSS), exfoliative skin diseases in which the outer layer of epidermis gets separated from the underlying tissues. u Staphylococcal Scalded Skin Syndrome u Bullous exfoliative dermatitis that occurs primarily in newborns and previously healthy young children u Severe form: Ritter’s Disease u Milder form: Pemphigus neonatorum and bullous impetigo (localized form) Laboratory identification of Staphylococcus and Micrococcus  ISOLATION AND IDENTIFICATION u Grow easily on routine laboratory culture media – sheep blood agar (SBA) u Selective medium (for heavily contaminated specimen) u Mannitol Salt Agar (MSA), Columbia Colistin–nalidixic Acid Agar (CNA), or Phenylethyl Alcohol (PEA) agar u High NaCl concentration (7.5%) in MSA makes this medium selective for Staphylococcus, u Incorporation of mannitol and phenol red distinguishes S. aureus from most CoNS. A. Microscopic Examination u Purulent exudates, joint fluids, aspirated secretions, and other body fluids – cocci and PMN easily seen when these sites are infected with staphylococci u Culture should be done regardless of the results of the microscopic examination – genus or species cannot be appropriately identified u Aspirate is the best sample, a single swab would be less satisfactory for both culture and smear results A. Microscopic Examination u Spherical cocci; arranged characteristically in grape-like clusters u May also be found singly, in pairs and in short chains of three or four cells, especially when examined from liquid culture. u Long chains never occur u Non spore forming, non motile and usually non capsulated with the exception of rare strains. u Stain readily with aniline dyes and are uniformly gram-positive B. Cultural Characteristics Staphylococci: u SBA (after 18-24 hours of incubation at 35° C to 37° C) - round, smooth, white, creamy colonies u BAP - produce hemolytic zones around the colonies (beta-hemolytic) u Rarely exhibit pigment production (yellow) with extended incubation Staphylococcus aureus: u Nutrient agar – 1–3 mm in diameter and have a smooth glistening surface, an entire edge, a soft butyrous consistency and an opaque, pigmented appearance u Most strains produce golden-yellow (aureus) pigment (staphyloxanthin) u White-colony strains of S. aureus are fully virulent. B. Cultural Characteristics Staphylococcus aureus: u Blood Agar u Colonies have the same appearances as on nutrient agar, but may be surrounded by a zone of ß-hemolysis u Hemolysis is more likely to be present if sheep, human or rabbit blood is used. u Phenolphthalein Phosphate Agar (PPA) q An indicator medium and assists in the identification of S. aureus in mixed cultures q Colonies is similar to those on nutrient agar § Become bright pink when culture plate is inverted over ammonia for a minute or so Staphylococcus aureus: u Selective Salt Media: 1. 7–10% of sodium chloride may be added to Nutrient Agar (Salt Agar) or Milk Agar (Salt Milk Agar) 2. Mannitol Salt Agar with 1% mannitol B. Cultural 3. 7.5% NaCl Characteristics 4. Nutrient Agar with Phenol Red 5. Ludlam’s Medium containing lithium chloride and tellurite 6. Salt Cooked Meat Broth (10% NaCl) u S. epidermidis – small- to medium-sized, nonhemolytic, gray-to-white colonies; some weakly hemolytic u S. saprophyticus – slightly larger colonies, with about 50% of the strains producing a yellow pigment u S. haemolyticus – medium sized colonies, with moderate or weak hemolysis and variable pigment production u S. lugdunensis – small to medium size colonies, often hemolytic u NOTE: Identification of Staphylococci on the basis of colony morphology alone should not be done.  C. Identification Methods 1. Catalase Test – demonstrate the presence of catalase à an enzyme that catalyses the release of oxygen from hydrogen peroxide (H2O2) u To distinguish Staphylococci spp. And Micrococci spp. (positive) u Reagent: 3% H2O2 u Positive result: bubble formation or effervescence B. Identification Methods 2. Coagulase (Slide or Tube Method) u Best single pathogenicity test for staphylococcus u 2 methods: 1. Slide Method (screening test) u Reagent: plasma u Detects bound coagulase (clumping factor) u Positive result: agglutination 2. Tube Method (confirmatory test) u Reagent : 0.5 mL rabbit’s plasma u Detects unbound coagulase (staphylocoagulase) u 35ºC incubation; initial time = 2 hours u 4 hours incubation (if negative, continue S. schleiferi incubation for 24 hours) u Positive result: coagulum/clot B. Identification Methods 3. Oxidation-Fermentation (O/F) Reactions u Medium used: O/F Glucose Medium u Two methods: 1. Open Method 2. Closed Method – add mineral oil as a barrier for oxygen u Interpretation of results: § Closed = (+) yellow: Fermentative § Open = (+) yellow; Oxidative § If both (+) = Staphylococci § If Closed is (-) and Open is (+) = Hugh and Leifson’s Medium Micrococcus  B. Identification Methods 4. Modified Oxidase Test Active Chemical component Tetra- methyl-para- phenylene diamine dihydrochloride Results: § (+) = blue to purple to black complex [Micrococci] § (-) = no color change [Staphylococcus] B. Identification Methods 5. Mannitol Fermentation Staphylococcus spp. Fermentative (Glucose) Medium: Mannitol Salt Agar (+) result: yellow [S. aureus] (-) result: pink [CONS] Phenol red B. Identification Methods DNA HYDROLYSIS TEST 6. DNAse Test Medium: DNAse Medium Incubate at 25ºC for several hours (+) colorless zone (+) = S. aureus (-) = S. epidermidis 7. Gelatin Liquefaction/Hydrolysis Test 12% Gelatin Medium (Tube) Stabbing method Incubate at 35ºC for 16-18 hours (+) Liquefaction è Refrigeration (30 mins) (+) = S. aureus (-) = S. epidermidis  B. Identification Methods 8. Novobiocin Susceptibility S. aureus S. epidermidis S. saprophyticus Coagulase + - - Novobiocin S S R Polymyxin R S S  9. Sugar Fermentation u Fermentsa range of sugars producing acid but no gas. u Sugar fermentation is of no B. Identification diagnostic value except for Methods mannitol, which is usually fermented anaerobically by S. aureus but not by other species Coagulase-negative Staphylococci (CONS) Staphylococcus epidermidis u Invariably present on normal man skin. u Infections are predominantly hospital acquired u Predisposing factors: uCatheterization uMedical Implantation uImmunosuppressive therapy u Most common cause of prosthetic valve endocarditis Staphylococcus saphrophyticus u UTIs in young women u Low numbers (