BACTE-M2-LEC.pdf

Staphylococcus aureus: Infection or Associated Disease A. Skin and Wound Infections Folliculitis and Furuncles - Mild inflammation of hair follicles; if if grows bigger it is called furuncles (boils/pigsa), a large raised superficial abscess Carbuncles...

Staphylococcus aureus: Infection or Associated Disease A. Skin and Wound Infections Folliculitis and Furuncles - Mild inflammation of hair follicles; if if grows bigger it is called furuncles (boils/pigsa), a large raised superficial abscess Carbuncles - invasive lesions develop from multiple furuncles and may progress into bigger tissues Bullous Impetigo - pustules that are larger and surrounded by erythema Toxin Mediated Infection Scalded Skin Syndrome - (Ritter's disease) extensive exfoliation or dermatitis caused by epidermolytic toxin (Staphylococcal Exfoliative Toxin) Toxic Shock Syndrome - (Fatal) Chills, Fever, Vomiting, Diarrhea, Muscle Aches, and Rashes that lead to hypotension (LOW BP) or shock Toxic Epidermal Necrolysis - Drug Induced Staphylococci B. Staphylococcus epidermidis (Common; Coagulase Negative Staphylococci i. Virulence Factor - Exopolysaccharide Slime or Biofilm (enhances the adhesion of slime layer to organs/inanimate objects such as teeth, catheter, or medical devices ii. Infections and Associated Diseases - ( Coagulase Negative Staphylococci) Nosocomial UTI, Prosthetic heart valve endocarditis C. Staphylococcus saprophyticus i. Virulence Factor - Adherence to epithelial cells (urogenital tract) & (Catheter) ii. Infections and Associated Diseases - UTI (sexually active young female patients & older woman in indwelling catheters); Honeymoon cystitis D. Staphylococcus lugdunensis - Coagulase negative (can give (+) slide, but tube negative) i. Virulence Factor - Gene mec A - encodes for oxacillin resistance Infections and Associated Diseases - community associated & hospital associates (causes endocarditis and UTI) E. Staphylococcus haemolyticus i. Virulence Factor - Vancomycin resistance (ANTIBIOTIC) ii. Inspections and Associated Diseases - Community and Hospital acquired endocarditis and UTI Laboratory diagnosis for Staphylococci Isolation and Identification Cultural Characteristics S. aureus - Medium to large; round, smooth, entire, translucent, creamy; mostly pigmented yellow and is Beta-hemolytic; - old sock odor - butyrous S. epidermidis - Small to medium; translucent, gray-white colonies, non hemolytic S. saprophyticus - Large; smooth, opaques, conves, usually white, yellow or orange Identification Methods Biochemical tests for the detection of staphylococcus spp. 1. Bacitracin Susceptibility (Taxo A) Test for the susceptibility to P.04U Bacitracin 24hrs incubation Susceptible: Reaction of drug: Presence of zone inhibition; >10mm; Micrococcus Resistant: No zone of inhibition; Staphylococcus Micrococcus and Staphylococcus (+) catalase test Coagulase Test Test for the ability of bacteria to convert fibrinogen into fibrin Differentiate Staphylococcus aureus from coagulase negative Staphylococci; Example: S. epidermidis & S. saprophyticus 2 types: 1. Bound Coagulase - (Slide Method) 2. Free/Extracellular Coagulase - (Tube Testing) Coagulase Slide Test: Bound Coagulase Detects bound coagulase “clumping factor” Add a drop of NSS + Rabbit’s Plasma, observe for clump/clot formation Positive: Agglutination/Clump (Macroscopic Clumping); White fibrin clot. Example: S. aureus, S. lugdunensis, S. schleiferi Negative: No clumping (Smooth milky suspension). Example: S. epidermidis, and others Coagulase Slide Test: Free Coagulase Detects free coagulase (staphylocoagulase) NSS + Rabbit’s plasma; incubate for 4 hrs at 37 C = If 4hrs no clot, extend incubation at room temperature for 16hrs Total incubation time: 20 hrs ONLY Positive: Clot of any size. Example: S. aureus Negative: No clot even after 16-20hrs. Example: S. epidermidis & others 2. Growth in Colistin-Nalidixic Agar (CNA)/Phenylethyl Alcohol Agar (PEA) Selective for gram-positive bacteria Growth: Staphylococcus and Streptococcus No Growth: Total Inhibition: E. coli & Partial inhibition: P. mirabilis 3.Dnase Test Test the ability of the organism to hydrolyze DNA Media: DNAse Agar 2 Methods: ○ HCl Precipitation Method - inoculate at dnase agar, incubate, add 0.1M of HCL to colonies ○ (+) result: Clearing of agar around colonies ○ Dye Method: Methyl green/toluidine blue as an indicator ○ Methyl Green (+) result: Clear zone around colonies ○ Toluidine Blue (+) result: Pink zone around colonies Positive: Depends on the method; usually S. aureus Negative: No clearing; S. epidermidis &othere Example: Moraxella (g-cocci) & Serratia (g-bacilli) 4. Growth in Mannitol Salt Agar Selective and differential medium Test for the ability to ferment mannitol ○ Tolerate growth with the NaCl (Selective) ○ Test for the ability to ferment mannitol (Differential) Contains NaCl (7.5%) (Inhibiting agent), D-mannitol (sugar), phenol red (Color Indicator) Growth with fermentation: Yellow halos surrounding growth. (S. aureus) Growth without fermentation: Plate remains pink to red. (S. epidermidis) 5. Novobiocin Susceptibility Test for susceptibility to 5ug Novobiocin For CONS (S. epidermidis & S. saprophyticus) Susceptible: Zone of inhibition/diameter of >16mm; S. epidermidis  Resistance: 24hrs - dome shaped colonies/umbonate - >48hrs - crater/nail head appearance colonies/checker appearance - Capnophilic - Bile solubility Positive Virulence Factor - Capsular polysaccharide - Antigenic/antiphagocytic - Test: Neufeld Quellung Test/Capsular swelling test  Associated Disease - Pneumonia (lobar, community acquired pneumonia); rust colored sputum - Sinusitis, Otitis Media, Bacteremia Regarded as the #1 cause of Adult Bacterial Meningitis (5-29 y/o) D. Viridans Streptococci (A, C, F, G, N) - MAJOR THROAT FLORA Virulence Factor - Polysaccharide capsule, extracellular dextran and cell surface-associated proteins (adhesins-initiate attachment to host cells) Associated Disease - Subacute bacterial endocarditis (patients w damaged heart valves - Bacteremia, septicemia and cavities E. Strep-Like Organisms: NUTRITIONALLY VARIANT STREPTOCOCCI Pyridoxal Requiring (B6), Satelliting Streptococci, Thiol Requiring Streptococci Viridans Strep Vitamin B6 in order to grow Abiotrophia & Granulicatella (S. adjacens and S. defectives) F. Group D Streptococcus: Non Enterococci Non-Enterococci: (S. bovis/S. gallolyticus group) Group Enterococcus & Non Enterococcus are both positive in Bile Esculin Test Species: S. equinus, S. gallolyticus, S. infantarius, S. galactolyticus Encountered in blood cultures of patients with bacteremia, septicemia and endocarditis F. Group D Streptococcus: Enterococci Enterococcus Species: E. faecalis, E. faecium, E. durans, E. avium (+) PYR and can resist 6.05% NaCl Virulence Factor All are common for ADHESION - Extracellular surface protein - Serine protease - gelatinase - Cytolysins - Resistance to antibiotics/antimicrobial agents Associated Disease - Nosocomial infection - UTI (catheterization), Bacteremia (hemodialysis & surgery), endocarditis (prosthetic heart valves) LABORATORY TESTS FOR IDENTIFICATION OF STREPTOCOCCI 1. Bacitracin Susceptibility test (Taxo A) Positive = Group A (Susceptible), ZOI Negative = Group B (Resistant) 2. Sulfamethoxazole-Trimethoprim Susceptibility Test for susceptibility to 1.25 ug of SXT disk Positive: Susceptible, ZOI Negative: Resistant, no ZOI; Group A (S. pyogenes) Group B (S. galactiae) 3. CAMP Reaction Test for the synergistic hemolysis between group B Streptococcus and B-hemolytic S. aureus Enhancement of beta hemolysis CAMP - diffusible protein like compound produced only by Group B streptococci/Streptococcus agalactiae Positive: Enhanced hemolysis, arrow head pattern Negative: No zone of beta hemolysis 4. Hippurate Hydrolysis Ability of organism to hydrolyze hippurate acid (substrate) to benzoic acid and glycine (end product) Glycine detected w/ NINHYDRIN reagent Purple color =glycine Positive: Deep blue-purple color (Group B S. agalactiae) Negative: Colorless result (Group A) 5. PYR Test Ability of organisms to hydrolyze L. pyrolidonyl - B-naphthylamide (PYR) = substrate/reagent Color Developer: DMCA Positive: Bright pink to red color w/in 5 mins (Group A) Negative: No color change 6. LAP Test Hydrolyze leucine - B-naphthylamide (Substrate) by enzyme leucine aminopeptidase to B-naphthylamide (End product) Color Developer: DMACA - cinnamaldehyde Positive: Development of red color within 1 min Negative: No color change 7. Bile Esculin Hydrolysis to grow in the presence of 40% bile and hydrolyze esculin Positive: Blackening of agar (Group D Streptococci) Negative: No Blackening of agar Media: Bile Esculin Agar 8. Salt Tolerance Test (6.5% NaCl) Test the ability of organism to grow in 6.5% NaCl Positive: Visible turbidity in broth (+) GROWTH; (Group D Enterococcus) Negative: Clear; Lack of Growth; (Group D Non Enterococcus) 9. Optochin (Taxo P) Susceptibility - Ethylenehydrocupreine hydrochloride Used to differentiate Streptococcus pneumoniae from other a-hemolytic streptococci (Viridans) Positive: ZOI (>14mm susceptible); S. pneumoniae Negative: No ZOI (