WEEK 7 - Staphylococcus.pdf

BACT211: CLINICAL BACTERIOLOGY WEEK 5 STAPHYLOCOCCI 2nd SEMESTER | S.Y. 2021-2022 LECTURER: MIKHAIL VALDESCNA CHARACTERISTICS o Colonies: ye...

BACT211: CLINICAL BACTERIOLOGY WEEK 5 STAPHYLOCOCCI 2nd SEMESTER | S.Y. 2021-2022 LECTURER: MIKHAIL VALDESCNA CHARACTERISTICS o Colonies: yellow pigment Family: Staphylococcaceae Rothia mucilaginosa Catalase (+) Aerococcus Coagulase (+) & some are Coagulase (-) CoNS Alloiococcus otits Gram (+) cocci in singly, in pirs and in clusters Resembles the family: Micrococcaceae; genus: Micrococcus o Catalase (+) o Coagulase (-) o Gram (+) cocci in pairs, tetrads, and, ultimately, irregular clusters o Found in environment and human skin Figure. 14.1 Micrococci growing on sheep blood agar showing yellow pigment TABLE 14.1 Differentiation Between Staphylococci and Micrococci in the Routine Laboratory (Mahon) Test Staphylococci Micrococci Modified oxidase (Microdase) - + Anaerobic acid production from Except M. glucose kristinae + and M. varians Anaerobic acid production from glycerol in the presence of + - erythromycin Growth on Furoxone-Tween 80-oil - + red O agar Resistance to bacitracin (0.04 U) 𝑅∗𝑐 S Resistance lysosome R S Resistance to lysostaphin (200 𝑆 ∗𝑐 R ug/mL) *R= resistant; s=sensitive *Micrococcus kristinae and Micrococcus varians are positive (Mahon) * 𝑅, 𝑆 𝑐 some stains show opposite reaction. (Mahon) Test Staphylococci Micrococci Rothia Aerococcus Enterococcus Except S. sciuri, Catalase + ± - - Macrococcus caseolyticus, S. lentus & Modified oxidase + - - - S. vitulus Except M. Aerotolerance FA kristinae and M. FA FA FA varians Resistance to bacitracin R S R/S S R (0.04 U) Resistance to furazolidone S R R/S S S (100 ug) Resistance to lysostaphin S R R R R (200 ug/mL) T A B I O S A S|1  BACT211: CLINICAL BACTERIOLOGY WEEK 5 STAPHYLOCOCCI 2nd SEMESTER | S.Y. 2021-2022 LECTURER: MIKHAIL VALDESCNA STAPHYLOCOCCI Folliculitis Mild inflammation of a hair follicle or oil gland; the Staphle meaning “bunches of grapes” infected area is raised and Nonmotile, non-spore-forming, and aerobic or red facultatively anaerobic EXCEPT S. saccharolyticus (obligate anaerobe) Normal flora of skin and mucous membranes of human & animals Colonies: medium sized (4 to 8 mm) and appear cream-colored, white or rarely light gold, and “butterfly-looking” Furuncles (boils) Fastidious strains requirements: CO2 hemin, or extension of menadione with at least 48 hrs of incubation folliculitis o Hemin- Factor X are large o Menadione- Vitamin K3 superficial abscesses Carbuncles Occur when larger More invasive PATHOLOGY lesions develop from multiple Organisms Infections furuncles, which S. aureus Cutaneous can progress into infections deeper tissues (folliculitis, With fever, chills, furuncles, indicating systemic carbuncles & spread bullous impetigo) Food poisoning Scalded skin syndrome (SSS) Toxic shock syndrome (TSS) Toxic epidermal Bullous impetigo Caused by S. aureus differs necrolysis (TEN) from streptococcal nonbullous impetigo in that S. epidermis Nosocomial staphylococcal pustule are infections large and surrounded by a S. saprophyticus UTI (in adolescent small zone of erythema girl and young women) S. haemolyticus Wound Septicemia UTIs Native valve infections S. lugdunensis Catheter-related bacteremia and endocarditis STAPHYLOCOCCUS AUREUS T A B I O S A S|2  WEEK 5: STAPHYLOCOCCUS Six Virulence Factors δ-hemolysin: less toxic than α-hemolysin or β- hemolysin 1. ENTEROTOXINS γ-hemolysin: (PVL) Panton-Valentine leucocidin Groups A-E & G-J Serologically identified Staphylococcal leucocidin Staphylococcal food A, B,D o (PVL) Panton-Valentine leucocidin poisoning Reheating o Exotoxin lethal to PMN contaminated food o Suppresses phagocytosis does not prevent o Associated with severe cutaneous infections disease and necrotizing pneumonia TSS (toxic shock syndrome) B, C, G, I o Often associated with community-acquired Staphylococcal staphylococcal infections pseudomembranous B enterocolitis Heat stable exotoxins (100℃ for 30 mins) Interacting with TSST-1: Activating an aggressive, interact with T cells over reactive immune response 2. Toxic Shock Syndrome Toxin-1 5. Enzymes TSS Previously known as enterotoxin F Coagulase, protease, hyaluronidase & lipase Super-antigen stimulating T-cell proliferation o Staphylocoagulase Production of large amount of cytokines for the ▪ S. aureus symptoms o Hyalurodinase Low concentrations: causes leakage by endothelial ▪ S. aureus cells ▪ Hydrolyzes hyaluronic acid present in the intracellular ground substance Higher concentrations: cytotoxic that makes up connective tissues, 3. Exfoliative Toxin permitting the spread of bacteria during infection Epidermolytic toxin o Lipase Staphylococcal SSS; Ritter disease ▪ By both coagulase (+) and CoNS Bullous impetigo ▪ Act on lipids present on the surface of the skin 6. Protein A Cellular components in cell wall of S. aureus Bind the Fc portion of immunoglobulin G (IgG) o Block phagocytosis and inhibit action of IgG Hemolysins α Damages erythrocytes, 4. Cytolytic Toxin platelets, and macrophages Extracellular proteins that affect red blood cells and β also known as leukocytes sphingomyelinase C, Lysins and Leukocidins disrupts the erythrocyte S. aureus: α, β, γ, δ hemolysis plasma membranes. o α-hemolysin: lyse RBC, damage platelets and Responsible for CAMP assay effectiveness macrophages and causes severe tissue δ Found in some CoNS strains damage as well as S. aureus. Less o β-hemolysin: also known as toxic than other hemolysins Sphingomyelinase C or “hot-cold lysin” γ Associated with Panton- ▪ acts on sphingomyelin in the Valentine leukocidin plasma membrane of erythrocytes Panton-Valentine Polymorphonuclear ▪ act in CAMP test (Christie, Atkins, Leukocidin leukocyte toxicity and Munch-Petersen) β -Lactamase Enzyme that cleaves the ring enhanced hemolytic activity on incubation at 37℃ and structure of penicillins and subsequent exposure to cold (4℃) derivative antibiotics making them ineffective T A B I O S A S|3  WEEK 5: STAPHYLOCOCCUS Hyaluronidase Permits bacteria to spread Culture through connective tissues SBA, MSA, CAN, PEA, CHROMagar Staph Lipases Common to S. aureus and aureus CoNS. Degrades lipids on 18 to 24 hours of incubation at 35℃ to 37℃ skin surface making it more susceptible to bacterial entry into epidermal layers Staphylocoagulase Responsible for a positive tube coagulase test result. Also present in S. intermedius, S. pseudintermedius, S.hycius, S. delphini, S. lutrae, S FIG. 14.4 Staphylococcus aureus growing on sheep blood agar showing β-hemolytic, creamy, buttery-looking colonies (Mahon) agnetis , and some S. schleiferi Toxic shock syndrome toxin- A superantigen causing an 1 overreactive immune response. Formerly known as enterotoxin F. Protein A Binds IgG and prevents phagocytosis Enterotoxins A–E, G, J Enterotoxins A, B, and D are FIG. 14.5 Coagulase-negative staphylococci growing on sheep blood agar, revealing the cause of the majority of nonhemolytic, white creamy colonies (Mahon) staphylococcal food Macroscopic Examination poisoning cases; heat stable. Enterotoxins B and C, and Colonies: rarely G and I, can cause o S. aureus: round, smooth, white, creamy enterocolitis colonies on SBA Exfoliative toxins Also known as epidermolytic o S. epidermis: small-to medium-sized, toxin. Types A and B. Solely nonhemolytic, gray-to-white colonies responsible for SSS and present in a minority of S. aureus species. May also cause bullous impetigo CAMP, Christie, Atkins, and Munch-Petersen; CoNS, coagulase-negative staphylococcal; IgG, immunoglobulin G; SSS, scalded skin syndrome. Mahon, C. et al. (2019). Textbook Of Diagnostic Microbiology (6th ed.) FIG. 14.3 Microscopic morphology of Staphylococcus spp. On Gram stain, gram- WORKFLOW negative–looking cells show how older cells decolorize (×1000). Specimen Collection Macroscopic Examination From the site of infection with aseptic technique Gram (+) cocci in singly, in pairs and in clusters No special procedures MANNITOL SALT AGAR Aspirates: ideal sample 7.5% NaCl (mahon) or 10% baileys in MSA: selective Swabs: less satisfactory for both culture and smear for S. aureus results Ferment mannitol; phenol red as pH indicator Forming: colonies surrounded by yellow halo Direct Microscopic Examination S. saprophyticus: may also ferment mannitol; may resemble Gram (+) cocci with PMN cells colonies of S. aureus May produce hemolytic zones around the colonies FIG. 14.2 Numerous gram-positive cocci in clusters, with many polymorphonuclear cells from an aspirated abscess in staphylococcal disease (Gram stain, original magnification ×1000) (Mahon) SBA- Sheep’s Blood Agar MSA- Mannitol Salt Agar CAN- Colistin and Nalidixic Agar PEA- Phenylethyl Alcohol Agar T A B I O S A S|4  WEEK 5: STAPHYLOCOCCUS Principle: catalase mediates the breakdown of hydrogen peroxide (30%0 H2O2) into oxygen and water Result: o (+) bubble formation; staphylococci and micrococci o (-) no or few bubble formation Microdase Test (aka Modified Oxidase Test) Principle: oxidase enzyme reacts with the oxidase reagent and cytochrome C to form the colored compound, indophenol after 2 mins Result: Biochemical Test o (+) development of blue to purple-blue color; Catalase micrococci o (-) no color o change; staphylococci FIG 13-27. Microdase Tesr. A= Positive; B=Negative Bacitracin Test Oxidation-Fermentation (O/F) Reactions Principle: determine the effect of a small amount of bacitracin (0.04U) on an organism Principle: determine whether an organism uses Result: carbohydrate substrates to produce acid by products o (+) presence of zone of inhibition around the using OF glucose medium disk o Glucose, xylose, mannitol, lactose, sucrose, o (-) no zone of inhibition and maltose Result: o (+) yellow medium; acid production: staphylococci ▪ S. saprophyticus ▪ S. auricularis ▪ S. hominis ▪ S. xylosus ▪ S. cohnii o Mixed acids (lactic acid, propionic acid & o (-) no change in color; no acid production: succinic acid) micrococci Oxidation ▪ Except M. kristinae and M. varians o Glucose (glycolysis) → Pyruvic Acid → CO2 Fermentation o Requires oxygen (aerobic respiration) o Glucose (glycolysis) → Pyruvic Acid; end o Inorganic molecule (anaerobic respiration) product: acid or acid with gas o Single acid (homolactic acid fermenters T A B I O S A S|5  WEEK 5: STAPHYLOCOCCUS Asaccharolytic: not use any CHO instead use other organic molecules such as AA energy and carbon source Sterile melted petrolatum or sterile paraffin oil Bromthymol blue: green (uninoculated) yellow (acid) blue (alkaline) 2 forms of coagulase enzyme: Bound coagulase/ clumping factor = Slide Coagulase Test If results are suggesting that the genus is staphylococci proceed to… Bound to the bacterial cell wall and reacts directly with fibrinogen in plasma Coagulate Test o Human, rabbit, or pig plasma in EDTA Principle: used to differentiate Staphylococcus aureus Alternation of fibrinogen → precipitates on from CoNS by detecting enzyme coagulase the staphylococcal cell Result: Fibrin clot (clumping of cells); (+) S. aureus, o (+) fibrin clot = S. aureus S. lugdunensis and S. schleiferi o (-) no fibrin clot = CoNS Free coagulase/ Staphylocoagulase = Tube Coagulate Test 5 or 10-15% of S. aureus organisms do not produce clumping factor, any negative slide coagulase test result must be confirmed with the tube method Extracellular protein enzyme (free coagulase) + CRF (coagulase-reacting factor) Coagulase-CRF complex + fibrinogen Fibrin clot; possibility of autolysis due to fibrinolysin T A B I O S A S|6  WEEK 5: STAPHYLOCOCCUS o Laboratory scientists should look for clot formation after 4-hours of incubation at 37℃. If no clot appears, the tube should be left at room temperature and checked the following day. Fibrinolysin activity is enhanced at 37℃. Pyrrolidonyl Arylamidase (PYR) Test Principle: differentiate S. aureus from other CoNS by the presence of the enzyme L- pyrroglutamylaminopeptidase L-pyrroglutamylaminopeptidase (hydrolyze) → L-pyrrolidonyl-β-naphthylamide/PYR (substrate) = β-naphthylamine β-naphthylamine + N, N- methylaminocinnamaldehyde/p- dimethylaminocinnaldehyde (reagent) Result: o (+) bright red color = S. lugdunesis, S. intermedius, S. schleiferi o (-) no color change or an orange color = S. aureus Vogue-Proskauer (VP) Test Principle: determine the ability of some organisms to MRSA (methicillin-resistant Staphylococcus aureus) produce neutral end products from glucose fermentation = acetyl-methylcarbinol or acetoin Penicillin-resistant (β-lactam) strains of S. aureus due to presence of the mecA gene Result: o (+) red color; S. aureus Requires penicillinase-resistant penicillins, such as o (-) yellow color; S. intermedius, S. nafcillin, oxacillin, or cefoxitin lugdunesis, S. haemolyticus & S. schleiferi Types: o CA-MRSA, HA-MRSA, MRSA MRSE TOC: Vancomycin VISA (vancomycin-intermediate Staphylococcus aureus) VRSA (vancomycin-resistant Staphylococcus aureus) Macrolide Resistance Resistance to clindamycin Modified double disk diffusion test (D-zone test) Novobiocin Susceptibility Test Principle: determine the effect of a 5-𝜇g novobiocin disk on an organisn Result: o (+) presence of zone of inhibition around the disk; other CoNS o (-) no zone of inhibition; S. saprophyticus T A B I O S A S|7