MICR 111 3MT03 Midterms PDF

MICR_111 NATHANIEL VERON M. CASAMINA, MSMT, RMT, ASCP | 3MT03 3RD YEAR, 1ST SEMESTER BY: MANALO, A. & MILAR, K. [TRANS] LESSON...

MICR_111 NATHANIEL VERON M. CASAMINA, MSMT, RMT, ASCP | 3MT03 3RD YEAR, 1ST SEMESTER BY: MANALO, A. & MILAR, K. [TRANS] LESSON #6:STAPHYLOCOCCI OUTLINE COAGULASE NEGATIVE STAPHYLOCOCCI S. epidermidis group I. Staphylococci S. saprophyticus group II. Staphylococcus Aureus S. simulans group III. Schematic Diagram for Identification of S. intermedius group Staphylococci S. sciuri group IV. Other Laboratory Tests S. hyicus group Unspecified group o S. lugdunensis STAPHYLOCOCCI ▪ When you perform coagulase test, they Derived from the Greek words “staphule”, which means will produce a negative result in the tube “bunches of grapes”, and kokkos, which means coagulase test. “berries” Gram-positive cocci that belong to the family MICROCOCCUS Staphylococcaceae. Different Micrococcus from Staphylococci Cataase-producing and facultatively anaerobic bacteria Part of the normal flora of the skin. except S. saccharolyticus, which is an obligate anaerobe. Gram Stain: Normal inhabitants of the skin, mucous membranes, and o Catalase + intestines. o Oxidase + Species which are associated with human infections are o Coagulase - colonizers of various skin and mucosal surfaces. Differentiate Micrococcus to Coagulase Negative Staphylococcus, we perform Oxidase Test/Microdase Test. GENERAL CHARACTERISTICS Presence of purple colored reaction after addition of Nonmotile, non spore forming, aerobic, facultative, Kovacs reagent and colony of organism indicates positive obligate anaerobe. result for oxidase. Meaning this organism possesses the o Colony: Pin head, cream-colored, light gold, and enzyme oxidase. buttery looking ▪ Blood Agar Plate/ BAP- Primary plated media for microbial identification o Gram Stain ▪ Gram + cocci (spherical, clustered) ▪ Grape-like appearance o Catalase - Differentiate Staph (+) vs Strep (-) ▪ All are catalase positive ▪ Streptococci are arrange in chains = catalase negative ▪ Presence of bubble formation indicates Table No. 1 Differentiation Tets positive reaction DEFINITION TESTS o Coagulase - Differentiate S. aureus (+) vs CoNS Differentiate Staphylococcus from Perform Catalase Test (-) Streptococcus ▪ Presence of clot formation is the positive Differentiate CoNS from Perform Coagulase Test result for coagulase and indicates S. Staphylococcus aureus aureus species. Differentiate Micrococcus from CoNS Perfrom Oxidase Test and ▪ Two methods: Perfrom Bacitracin Test 1. Slide Coagulase - Detects clumping factor, a presumptive test. Table No. 2 Differential Tests between Staphyloccoci and Microccoci TEST RESULT QUALITY 2. Tube Coagulase - Detects true CONTROL coagulase factor Bacitracin/ Taco A (0.04 Susceptible - Susceptible: units) Micrococci Streptococcus Medium: BAP or MHA (≥ 10 mm zone of pyogenes ATCC inhibition) 19615 Resistant - Micrococcus luteus Staphylococci ATCC 10240 (≤ 6 mm or no zone of inhibition) Resistant: Streptococcus agalactiae ATCC 27956 1 TRINITY UNIVERSITY OF ASIA – COLLEGE OF MEDICAL TECHNOLOGY | 3MT03 LESSON #6: STAPHYLOCOCCI Furazolidone Susceptible - Susceptible: Table No. 3 CoNS versus Micrococci Susceptibility (100 -μg) Staphylococci Staphylococcus Medium: BAP (≥ 15 mm zone of aureus ATCC TEST STAPHYLOCOCCI MICROCOCCI inhibition) 25923 Modified oxidase - + Resistant - Resistant: Micrococci Micrococcus luteus Anaerobic acid production + -ᵇ (≤ 9 mm or no ATCC 10240 from glucose zone of inhibition) Lysostaphin Susceptible - Susceptible: Growth on Furoxone- Tween - + Suscpetibility Staphylococci Staphylococcus 80- oil red O agar (1 -μg or 200g/L) (10mm - 16mm aureus ATCC Medium: MHA or broth zone of inhibition) 25923 Anaerobic acid production + - Resistant - Resistant: from glycerol in the Micrococci Micrococcus luteus presence of erthremyein ATCC 4698 Modified Positive - Blue Positive : Oxidase/Microdase color within 2 mins Micrococcus luteus Resistance to bacitracin Rᶜ S - Reagent: ATCC 10240 (0.04 units) Tetramethyl-p- Negative - No phenylenedia color change Negative: Lysosome (50-mg disk) R S mine in Staphylococcus dimethyl aureus ATCC sulfoxide 25923 Lysostaphin test Sᶜ R Growth on Positive - Postive: Furoxone-Tween-80-Oil Micrococci Micrococcus luteus R, resistant; S, sensitive Red O Agar ATCC 10240 ᵃCommercially available, useful for presumptive identification Negative - ᵇMicrococcus kristinae and Micrococcus varians are positive. Staphylococci Negative: ᶜSome stains show opposite reaction Staphylococcus aureus ATCC Modified from Schumacher-Perdreau F: Clinical significance and 25923 laboratory diagnosis of CoNS, Clin Microbiol News 13:97, 1991. Anaerobic Acid Positive: Production from Staphylococci Bacitracin and Lysostaphin test are inversely proportional. Carbohydrates Open and Closed Medium: Baird Parker’s Tubes : Yellow SMB-RL Modification OF 1. First, perform oxidase test to differentiate CoNs from Microcci Negative: 2. Next perform a bacitracin test, a susceptible reaction favors Glucose: 1% Micrococci your micrococci. Closed/Anaerobic pH indicator: Bromcresol Tube: Purple / No purple color change (but CONS CLINICALLY SIGNIFICANT SPECIES in open/aerobic S. epidermidis (Nosocomial Acquired Infection/ Implanted Note: Sometimes it is tube it is yellow Medical Devices, Endocarditis) called color) Oxidation-Fermentation S. saprophyticus (Urinary Tract Infection, Cystitis) Test S. lugdunensis (Catheter Related Bacteremia and Anaerobic Acid Positive/ Yellow Infective Endocarditis) Production from color - S. haemolyticus (occasionally recovered from Native Glycerol-Erthromycin Staphylococci Valve Infection, Septicemia, and UTI) Medium Erthromycin: (4-μg) Negative - pH indicator: Bromcresol Micrococci purple STAPHYLOCOCCUS AUREUS Most clinically significant species True coagulase-positive and most virulent Principle Virulence Factor: Coagulase Halophilic: 7.5% to 10% NaCL (Mannitol Salt Agar) Colony on BAP: Golden yellow color with narrow zone of beta-hemolytic pattern (sometimes no hemolysis) 2 TRINITY UNIVERSITY OF ASIA – COLLEGE OF MEDICAL TECHNOLOGY | 3MT03 LESSON #6: STAPHYLOCOCCI Chiefly responsible for the various skin, wound, and deep Food Poisoning tissue infections. Pseudomembranous enterocolitis This organism causes infection when it enter a normally o Mostly associated with clostridium difficile sterile site (trauma or abrasion of the skin or mucosal infection surfaces). o There are also instances that staphylococcus Acquired through direct exposure with unwashed hands, aureus causes enterocolitis. It is possible as well as contact with inanimate objects. because of the enterotoxin A and B. It is also one of the common causes of food poisoning. Tampons Toxic Shock Syndrome (TSST-1) VIRULENCE FACTORS OF S. AUREUS o First identified in late 1970s and early 1980s Coagulase because of the use of absorbent tampons o “Coagulation” o Forgetting to replace tampons can cause toxic o Formation of fibrin clot shock syndrome o Protects the bacteria from phagocytosis o Symptoms: High fever or chills, Diarrhea, Hyaluronidase Headache, Low Blood Pressure, Nausea or o “Spreading factor” sickness, Rash, Seizures o The skin is very rich in Hyaluronic acid o Hyaluronic acid - a protective acid in the skin and that enzyme degrade it o Infection in S. aureus spreads easily because of Hyaluronidase Staphylokinase o Dissolves the fibrin clot o Hemolysin - can cause narrow zone of hemolysis in the blood agar Lipase o Dissolves the lipids allowing the entry of bacteria DNAse DIFFERENTIAL TEST FOR S. AUREUS o Destroys the DNA β-Lactamase 1. COAGULASE TEST o Responsible for resistance to penicillin and other Reagent: Rabbit plasma beta lactamase Anticoagulant: EDTA (Ethylenediaminetetraacetic acid) Slide Method (detects cell bond coagulase or clumping Table No. 4 Virulence Factors of S. aureus factor) ENZYMES TOXINS o + S. lugdunensis and S. schleiferi Coagulase Enterotoxin (Food poisoning) Tube Method (detects extracellular or free coagulase) - (A,B,C1,C2,D,E,G-J) o + S. hyicus, intermedius, lutrae, delphini and o A - Food poisoning schleiferi o B - Spoiled milk o B, C, G - Enterocolitis Table No. 5 Coagulase test Hyaluronidase Panton-Valentine Leukocidin SLIDE METHOD TUBE METHOD (Spread factor) (Cytolytic toxin) A colony of emulsified of is A tube containing 0.5mL of rabbit Staphylokinase Hemolysin emulsified in a drop of rabbit plasm is inoculated with colony and Lipase Exfoliatin A and B (SSS) plasma, then rotate the slide for then incubated at 35 degrees - Scalded Skin Syndrome (SSS) 30 secs. celsius for 1-4 hours. - Also known as Ritters Disease + Result: Clot formation within 30 + Result: Clot formation after DNAse Toxic Shock Syndrome Toxin 1 sec. incubation. β-Lactamase Protein A DISEASES AND INFECTIONS CAUSED BY S. AUREUS Abscesses and boils Cellulitis Folliculitis Furuncles Impetigo 2. MANNITOL FERMENTATION Cutaneous infection Culture medium: Mannitol Salt Agar Exfoliatin A and B + Result: Yellow Colored Colonies Scalded Skin Syndrome/Ritters Disease pH indicator: Phenol Red Enterotoxin A and B 3 TRINITY UNIVERSITY OF ASIA – COLLEGE OF MEDICAL TECHNOLOGY | 3MT03 LESSON #6: STAPHYLOCOCCI 3. DNASE TEST SCHEMATIC DIAGRAM FOR IDENTIFICATION OF Dnase Test (Dnase agar with methyl green dye and STAPHYLOCOCCI sodium chloride) 1. The doctor requests culture, and depending on the + Result: Clear zone around test organism specimens submitted, the primary thing to do is gram - Result: Dnase agar remain green in color stain and culture. 2. After gram stain, you discover that it is gram-positive cocci. 3. Wait for the growth of the colony BAP. You observe a creamy white colony, pin head colonies. 4. Differentiate staph and strep by performing catalase test, reagent: 3% hydrogen peroxide. 5. If it is catalase positive, then it is presumptive Staphylococci. If it is catalase positive, then it is presumptive Streptococcaea. 6. If it is catalase positive, perform coagulase testing to differentiate Staphylococcus aureus through coagulase-positive tube vs coagulase-negative 4. TELLURITE GLYCERINE AGAR TEST/ staphylococci. BAIRD-PARKER AGAR 7. 2 variants of coagulase-negative staphylococci. Jet black colonies of S. aureus a. Micrococcus spp. b. CoNS 8. If it is coagulase positive then it is Staphylococcus aureus. 9. If it is coagulase negative then perform oxidase/bacitracin susceptibility test. a. + oxidase (purple)/Susceptible to bacitracin: 5. POLYMYXIN SUSCEPTIBILITY TEST Micrococcus spp. Resistant (S. aureus) b. - oxidase (no change in color)/ Resistant to: CoNS MRSA 10. Perform Novobiocin susceptibility test. A type of S. aureus strain that is resistant to antibiotics a. 16: Sensitive Nasal swab is the common specimen used for detection 11. Resistant to Novobiocin = Staphylococcus for resistant strain saprophyticus Chromogenic/ CHROM agar test 12. Sensitive to Noobiocine = Staphylococcus o + result: pink to mauve color colonies after epidermidis 24-48 hours of incubation at 35 degrees celsius o mecA gene - encodes for PBP2a CLINICAL RELEVANCE - OTHER ANTIBIOTIC RESISTANCES Table No. 6 Antibiotic Abbreviations ABBREVIATIONS MEANING MSSA Methicillin-Sensitive S. aureus MRSA Methicillin-Resistant S. aureus VISA Vancomycin-Intermediate Sensitive S. aureus GISA Glycopeptide-Intermediate Sensitive S. aureus VRSA Vancomycin-Resistant S. aureus Table No. 7 Time Table ABBREVIATIONS 1956: Discovery Vancomycin 1958: Introduction of Vancomycin 1956: Discovery VISA 1956: Discovery VRSA → actual “reserve-antibiotic” is Linozoloid (last line of defense); only in severe cases → development of resistance must be restricted! 4 TRINITY UNIVERSITY OF ASIA – COLLEGE OF MEDICAL TECHNOLOGY | 3MT03