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    Cocci 2011 Cocci 2011 Presentation Transcript

    • Laboratory Diagnosis• Colonial Morphology (BAP) • S. aureus  Golden yellow colonies, smooth, entirely raised, - hemolytic (>24 hrs of incubation) • S. epidermidis  translucent, gray-white colonies, non- hemolytic • S. saprophyticus  (same as S. epidermidis)
    • Laboratory DiagnosisGram stain: Gram-positive cocciDifferentiatesStaphylococcus and Catalase testStreptococcusStaphylococcus Streptococcussp. sp.
    • Catalase test (Slide)Negative Positive
    • Medically important Staphylococci• Staphylococcus aureus• Staphylococcus epidermidis• Staphylococcus saprophyticus
    • Laboratory Diagnosis Catalase test Coagulase test/ MSAStaphylococcussp. S. aureus S. saprophyticus S. epidermidis
    • Coagulase Test• 2 types1.) Bound/Clumping factor  Slide test  (+) Result: agglutination of organism when mixed w/ plasma  not all strains of S. aureus produced clumping factor2.) Free  Tube test  (+) Clot formationAnticoagulant: EDTA
    • Coagulase test (Tube) Positive Negative
    • Mannitol Salt Agar• pH indicator: phenol red• Sugar: Mannitol• Salt conc.: 10%• (+) Result: Yellow Halo
    • Laboratory DiagnosisCoagulase test/ Novobiocin test MSA (S) (R)S. saprophyticusS. epidermidis S. epidermidis S. saprophyticus
    • Novobiocin disc testS. epidermidis S. saprophyticus
    • GENUS: Staphylococcus• Gram-positive• (0.5 – 1 m)• Non-motile, non-sporeforming• Catalase (+)• Facultative anaerobes
    • Medically important Staphylococci• Staphylococcus aureus• Staphylococcus epidermidis• Staphylococcus saprophyticus
    • Staphylococcus aureus• Catalase-positive, Coagulase-positive• Salt tolerant (7.5% NaCl)• Ferments mannitol on Mannitol salt agar
    • Reservoir• Normal flora on nasal mucosa and skin
    • Transmission• Spread via the hands and sneezing• Fomites• Surgical wounds• Lungs of cystic fibrosis patients• Foods associated with food poisoning (Ham/Canned meats, Custard pastries and potato salad)
    • Predisposing Factors for Infections• Any break in skin (sx)• Any foreign body (sx packing, sutures, tampons)• Ventilators• WBC <500/ L• Dse: CF, CGD• IV drug abuse
    • Virulence factors:A. Cell associated factorsB. Extracellular factors
    • A) CELL ASSOCIATED FACTORS:a) CELL ASSOCIATED POLYMERS 1. Cell wall polysaccharide (Peptidoglycan) 2. Teichoic acid 3. Capsular polysaccharide (some strains)b) CELL SURFACE PROTEINS: 1. Protein A (S. aureus) 2. Clumping factor (bound coagulase)- S. aureus
    • Staphylococcal cell wall
    • B) EXTRACELLULAR FACTORSa) Enzymes:1. Catalase – all Staph.2. Free coagulase (S. aureus)3. Lipase4. Hyaluronidase (Spreading factor)5. β-lactamases6. Staphylokinase (Fibrinolysin)7. Proteinases
    • B) EXTRACELLULAR FACTORSb) Toxins:1. Cytolytic toxins i) Hemolysins (α,β,γ,δ) ii) Leucocidin (Panton-Valentine toxin)2. Enterotoxin A-F (heat stable 60°C, 10 mins)3. Toxic shock syndrome toxin-1 (TSST-1)4. Exfoliative (epidermolytic toxin) .
    • Staphylococcal Diseases Infections Intoxications
    • 1) Skin and soft tissue infections:• Folliculitis, furuncle (boil), carbuncle, styes, abscess, wound infections, impetigo, paronychia and less often cellulitis.• Coagulase
    • 2) Musculoskeletal• Osteomyelitis, arthritis, bursitis, pyomyositis.
    • Osteomyelitis
    • 1. Respiratory: Tonsillitis, pharyngitis, sinusitis, otitis, bronchopneumonia, lung abscess, empyema, rarely pneumonia.2. Central nervous system: Abscess, meningitis, intracranial thrombophlebitis.3. Endovascular: Bacteremia, septicemia, pyemia, endocarditis.4. Urinary: Urinary tract infection.
    • Infective endocarditis (Acute)• Fever, malaise, leukocytosis, heart murmur (may be absent initially)• # 1 cause  S. aureus• Fibrin platelet mesh, cytolytic toxins
    • B) INTOXICATIOINS:The disease is caused by the bacterial exotoxins,which are produced either in the infected hostor preformed in vitro.There are 3 types-1. Food poisoning2. Toxic shock syndrome3. Staphylococcal scalded skin syndrome
    • Gastroenteritis (food poisoning)• 1-8 hours after ingesting toxin • Nausea • abdominal pain • Vomiting • followed by diarrhea • No fever• Enterotoxins A-F preformed in food (heat-stable)
    • Toxic Shock Syndrome (TSS) • High fever (abrupt) • Vomiting • Diarrhea • Myalgias • Scarlatiniform rash • Hypotension • Cardiac/Renal failure (severe cases) • TSST-1
    • Staphylococcal Scalded Skin Syndrome(SSSS)• Exfoliative toxin
    • Laboratory Diagnosis:Specimens collected: Depends on the type of infection.• Suppurative lesion- Pus,• Respiratory infection- Sputum,• Bacteremia & septicemia- Blood,• Food poisoning- Feces, vomit & the remains of suspected food,• For the detection of carriers- Nasal swab.
    • Treatment• Methicillin/ Nafcillin/ Oxacillin/ Cloxacillin• Methicillin-resistant S. aureus (MRSA) (due to changes in major penicillin-binding proteins) is commonly resistant to all antibiotics EXCEPT Vancomycin and Fusidic acid.• Topical mupirocin reduces nasal colonization.
    • Prevention• Basic hospital infection control
    • Staphylococcus epidermidis• Reservoir: skin and mucous membrane• Neonatal Sepsis• Peritonitis in patients with renal failure who are undergoing peritoneal dialysis through an indwelling catheter• Most common  CSF shunt infection• Infxn related to intravenous catheters and prosthetic implants (e.g., heart valves, vascular grafts, and joints)• Coagulase (-); Novobiocin (S)
    • Staphylococcus saprophyticus• Causes U.T.I., particularly in sexually active young women.• 2nd cause community acquired U.T.I. young women (Most common cause E. coli)Coagulase (-); Novobiocin (R)
    • Characteristics S.aureus S.epidermidis S.saprophyticusCoagulase + - -Novobiocin Sensitive Sensitive ResistantsensitivityAcid from + - -mannitolfermentationanaerobicallyHemolysis beta - (most) -
    • Laboratory DiagnosisGram stain: Gram-positive cocciDifferentiatesStaphylococcus and Catalase testStreptococcusStaphylococcus Streptococcussp. sp.
    • Important Streptococci• Streptococcus pyogenes• Streptococcus agalactiae• Enterococcus faecalis• Streptococcus bovis• Streptococcus pneumoniae• Viridans group
    • Streptococcus• Hemolysis varies by species:•••
    • Laboratory Diagnosis  OPTOCHIN BACITRACIN 6.5% NaCl (S)S. pyogenes (R)S. agalactiae(S)S.pneumoniae (+)Enterococcus(R)Viridans group ( - )S. bovis
    • Laboratory Diagnosis• Optochin “Taxo P” Disc test
    • Laboratory Diagnosis• Bacitracin “Taxo A” Disc test (S)
    • GENUS: Streptococcus • Gram-positive • Non-motile, non-sporeforming • Catalase (-) • Facultative anaerobes
    • Streptococci• Are serogrouped using known antibodies to the cell wall carbohydrates (Lancefield’s Group A-H, K-U)• Group A- Rhamnose-N-acetylglucosamine• Group B-Rhamnose-glucosamine polysaccharide• Group C-Rhamnose-N-acetylgalactosamine• Group D- Glycerol teichoic acid• Group F- Glucopyranosyl-N-acetylgalactosamine
    • Laboratory Diagnosis• Specimen Collection & Processing: • No special consideration; site• Antigen Detection • S. pyogenes  (throat) latex agglutination, Coagglutination, ELISA• Gram Stain
    • Laboratory Diagnosis• Cultivation • MOC: 5% Sheep’s Blood Agar (BAP)
    • Laboratory Diagnosis• Colonial appearance - Grayish white - Hemolysis
    • Streptococcus pyogenes (GABS)Distinguishing Characteristics Beta-hemolytic Group A Colonies inhibited by Bacitracin on BA Gram-positive cocci in chains Catalase-negative PYR (+)
    • Reservoir• Human throat• Skin
    • Transmission• Spread by respiratory droplets• Direct contact
    • Group A Streptococcal cell wall
    • Cell wall components• Hyaluronic acid capsule (a polysaccharide) is non- immunogenic; inhibits phagocytic uptake• M-protein: major virulence factor, hair-like projections; antiphagocytic, used to type group A Strep
    • Toxins• Hemolysins • Streptolysin O: immunogenic, hemolysin/cytolysin • Streptolysin S: non-immunogenic, hemolysin/cytolysin
    • Exotoxins A-C(pyrogenic/erythrogenic)• Phage-coded (e.g., the cells are lysogenized by a phage)• Cause fever and the rash of Scarlet fever• Inhibit liver clearance of endotoxin (from normal flora), creating shock-like conditions• Superantigens: activate many helper T cells by bridging T cell receptors and MHC class II markers without processed antigen
    • Spreading factors:• Streptokinase (fibrinolysin): breaks down fibrin clot• Streptococcal Dnase (Streptodornase): liquefies pus, extension of lesion• Hyaluronidase: hydrolyzes the ground substances of the connective tissues; important to spread in cellulitis
    • Diseases• Streptococcus pyogenes causes a wide variety of acute infections; some have immunologic sequelae
    • Acute (Suppurative) S.pyogenes Infxn • Pharyngitis (most common) • Scarlet fever • Pyoderma/ Impetigo (Also, cellulitis,necrotizing fasciitis (flesh-eating bacteria!), puerperal fever, lymphangitis, pneumonia, a toxic shock- like syndrome, etc.
    • Pharyngitis• Abrupt onset of sore throat• Fever• Malaise• Headache• Tonsillar abscesses• Tender anterior cervical lymph nodes• Lab: For Strep throat: Rapid antigen test (misses 25% of the strep throat); culture all “negatives”
    • Pyoderma/ Impetigo• Pus-producing skin infection (honey-crusted lesions)
    • Erysipelas• Brawny edema, advancing margin of infection
    • Necrotizing fasciitis• S. pyogenes  “flesh-eating bacteria”
    • Scarlet fever• Initial: S/Sx’s of pharyngitis• Followed by blanching, “sandpaper” rash
    • Scarlet fever• Pastia lines
    • Scarlet fever• Strawberry tongue
    • Non-suppurative Sequelae to GroupA Streptococcal Infections• Rheumatic fever• Acute glomerulonephritis (M12 serotype)
    • Rheumatic fever• Sequelae to : Pharyngitis with group A Strep (not group C)• Mechanism: in genetically susceptible individuals, the infection results in production of antibodies that cross- react with cardiac antigens
    • Rheumatic fever• Symptoms occurs 2-3 weeks after a pharyngeal infection• Lab: elevated ASO titers (>200)• Jones Criteria
    • Major Jones Criteria• “J NES”• J- Joints (Migratory arthritis)• -Carditis• N- Subcutaneous Nodules• E- Erythema marginatum• S- Sydenham chorea
    • Minor Jones Criteria• Fever• Arthralgias• Elevated acute phase reactants
    • Rheumatic fever• DIAGNOSIS: • 2 major or • 1 major & 2 minor
    • Acute Glomerulonephritis• Sequelae to: Pharyngitis or Cutaneous strep infxn• Mechanism: Immune complexes bound to glomeruli
    • Laboratory Diagnosis• PYR test• Principle:hydrolysis of L- pyrrolidonyl-- naphthylamide (PYR)
    • Treatment• Penicillin G  DOC• Beta-lactam drugs• Erythromycin
    • Prevention• Penicillin in RF px to prevent recurrent S. pyogenes pharyngitis
    • Streptococcus agalactiae = Group BStreptococci (GBS)• Distinguishing Characteristics • Beta-hemolytic • Bacitracin-resistant on BAP • Gram-positive cocci in chains • Group B • Catalase-negative, hydrolyzes hippurate • CAMP test-positive: CAMP(Christie-Atkins-Munch-Peterson) factor is a polypeptide that “compliments” a Staph aureus sphingomyelinase to make an area of new complete beta- hemolysis
    • Reservoir• Colonizes human vagina (15 – 20% of women)
    • Transmission• Newborn infected during birth• Increased risk with PROM
    • Pathogenesis• Beta-hemolysin
    • Diseases• Neonatal septicemia• Neonatal meningitis (Neonate – 2 mths)• Most common causative agent ( GEL) # 1 – S. agalactiae (GBS) 2 – E. coli Rare: L. monocytogenes
    • Laboratory Diagnosis• 0.04 U Bacitracin disk – Resistant• CAMP (Christie, Atkins, Munch- Peterson) Test  detects production of a diffusible, extracellular protein that enhaces hemolysis of sheep erythrocytes by S. aureus • (+) Arrowhead shape at the juncture of S. agalactiae & S. aureus
    • Treatment• Ampicillin with Cefotaxime or Gentamicin
    • Prevention• Treat mother prior to delivery if she had a previous baby with GBS, has documented GBS colonization, or prolonged rupture of membranes
    • Streptococcus pneumoniae(Pneumococcus)• Distinguishing Characteristics - Alpha-hemolytic - Colonies inhibited by optochin on BAP - Gram-positive, lancet-shaped diplococci (or short chains) - Lyse by bile- Quellung (+)
    • Reservoir• Human Upper Respiratory Tract
    • Transmission• Respiratory droplets; not considered highly communicable• Often colonizes without causing disease
    • Pathogenesis• IgA protease: colonization
    • Pathogenesis• Teichoic acids: attachment• Polysaccharide capsule: major virulence factor• Pneumolysin O: hemolysin/cytolysin • Damages respiratory epithelium (hemolysin similar to streptolysin O, which damages eukaryotic cells) • (Inhibits leukocyte respiratory burst and inhibits classical complement fixation.)
    • Pathogenesis• Pneumococcus in alveoli stimulate release of fluid and red and white cells producing “rusty sputum”• Peptidoglycan/ teichoic acids highly inflammatory in CNS
    • Diseases• Bacterial Pneumonia• Adult Meningitis• Otitis Media and Sinusitis in children• Septicemia
    • Bacterial Pneumonia• Most common bacterial cause, especially after 65 years but also in infants• Sx: • “big” shaking chills • Sharp pleural pain • High fever • Lobar with productive blood-tinged sputum (rusty-colored)
    • Predisposing Conditions forPneumonia• Antecedent influenza or measles infection: damage to mucociliary elevator• Chronic obstructive pulmonary disorders• Congestive heart failure• Alcoholism• Asplenia predisposes to septicemia
    • Adult Meningitis• Most common cause (> 40 y/o)• CSF: WBC (PMN) Glucose Protein Pressure
    • Otitis Media and Sinusitis in Children • Most common cause
    • Septicemia• In splenectomized patients
    • Treatment• Penicillin G  DOC• Resistance (both low level and high level) is chromosomal (altered penicillin-binding proteins); major concern in meningitis (Vancomycin  Rifampin used)
    • Prevention• Vaccine 23 serotypes of capsule
    • Viridans Streptococci (S. sanguis, S.mutans, etc.)• Distinguishing Characteristics • Alpha-hemolytic, resistant to optochin • Gram-positive cocci in chains • NOT bile soluble
    • Reservoir• Human oropharynx (normal flora)
    • Diseases• Dental carries• Infective Endocarditis (Subacute)
    • Dental carries• S. mutans dextran-mediated adherence glues oral flora onto teeth, forming plaque and causing caries
    • Infective Endocarditis• Sx: • Malaise • Fatigue • Anorexia • Night sweats • Weight loss• Predisposing factors: • Damage (or prosthetic) heart valve • Dental work w/o prophylactic antibiotics • Extremely poor oral hygiene
    • Pathogenesis• Dextran (biofilm)-mediated adherence onto tooth enamel or damaged heart valve and to each other (vegetation). Growth in vegetation protects organism from immune system.
    • Treatment• Penicillin G with Aminoglycoside for endocarditis
    • Prevention• For individuals with damage heart valve• Prophylactic penicillin prior to dental work
    • GENUS: Enterococcus• Catalase negative• PYR +• Hydrolyzes Esculin in 40% bile• (+) growth 6.5% NaCl
    • Enterococcus faecalis =Streptococcus faecalis• Distinguishing Characteristics • Group D Gram-positive cocci in chains • PYR test + • Catalase-negative, varied hemolysis • Hydrolyzes esculin in 40% bile (bile esculin agar turns black) • (+) growth 6.5% NaCl
    • Reservoir• Human colon• Urethra • Female genital tract
    • Pathogenesis/ PredisposingConditions • Bile/ Salt tolerance allows survival in bowel and gall bladder • During medical procedures on GI or GU tract: E. faecalis  bloodstream  previously damaged valves  ENDOCARDITIS (SBE)
    • Diseases• Urinary, biliary tract Infections• Infective endocarditis (SBE)
    • Treatment• All strains carry some drug resistance• Some vancomycin-resistant strains of Enterococcus faecium or E. faecalis: no reliably effective treatment
    • Prevention• Prophylactic use of penicillin and gentamicin in patients with damaged heart valves prior to intestinal or urinary tract manipulation
    • Gram-negative cocci
    • GENUS: Neisseria• Gram-negative• Diplococci with flattened sides• Oxidase positive
    • Important Genera• Neisseria meningitidis• Neisseria gonorrhoeae
    • NeisseriaSpecies N. meningitidis N. gonorrhoeaeCapsulePiliVaccinePortal of entry Respiratory GenitalGlucose UtilizationMaltose FermentationOxidase testBeta-lactamase prdxn Rare
    • Neisseria meningitidis(meningococcus)• Distinguishing Characteristics • Gram-negative kidney bean-shaped diplococci • Large capsule • Grows on chocolate (not blood) agar in 5-10% CO2 • Ferments maltose • Oxidase positive • 13 Serogroups: A, B, C, D,29E, H, I, K,L,X,Y,Z & W-135
    • Reservoir• Human nasopharyngeal area• ≥ 5% carriers (asymptomatic)
    • Transmission• Respiratory droplets• Oropharyngeal colonization• Spread to the meninges via the bloodstream• Disease occurs in only small percent of colonized
    • Pathogenesis• Important Virulence Factors • Polysaccharide capsule (most impt) • IgA protease allows oropharynx colonization • Endotoxin (LPS): fever, septic shock in meningococcemia, overproduction of outer membrane • Pili and outer membrane proteins important in ability to colonize and invade • Deficiency in late complement components (C5-8) predisposes to bacteremia
    • Diseases• Meningitis• Meningococcemia
    • Waterhouse-Friderichsen Syndrome• Most severe form of meningococcemia• High fever• Shock• DIC• Ecchymoses• Adrenal insufficiency• Coma• Death
    • Laboratory Diagnosis• Specimen: • Blood  culture only • CSF  smear, culture (tube #2), chemical determination • Petechial aspirate  smear/culture (?)
    • Laboratory Diagnosis• G/S• Presumptive dx: (+) gram-negative cocci on CSF smear• Presumptive Dx: (+) Oxidase test (tetramethyl-para- phenylenediamine- dihydrochloride)
    • Laboratory Diagnosis• Culture CAP 5-10% CO2 (candle jar) Incubate at 36-37 C at least 5 days before discarding as negative• Confirmatory test: Carbohydrate Fermentation test• (+) Glucose• (+) Maltose
    • Treatment• Penicillin G – DOC• Ceftriaxone• β- lactamase production (rare)
    • Prevention• Vaccine: capsular polysaccharide of strains Y, W-135, C, A
    • Prophylaxis• Rifampicin• Ciprofloxacin
    • Neisseria gonorrhoeae• Distinguishing Characteristics • Gram-negative kidney bean-shaped diplococci • Intracellular Gram-negative diplococci in PMNs from urethral smear is suggestive of N.g. • Sensitive to drying and cold
    • Reservoir• Human genital tract
    • Transmission• Sexual contact• Birth
    • Pathogenesis• Pili • Attachment to mucosal surfaces • Inhibit phagocytic uptake • Antigenic (immunogenic) variation • Most impt
    • Pathogenesis• Outer membrane Proteins • OMP I: Structural, antigen used in serotyping • OPA proteins (opacity): antigenic variation, adherence • IgA protease: aids in colonization and cellular uptake
    • Disease• Gonorrhea
    • Laboratory Diagnosis• Specimen • Discharge from the GUT • Discharge from the rectal mucosa • Discharge from the throat/ oropharynx • Skin lesions • Eye/ Conjuntival Discharge • Synovial Fluid
    • Laboratory Diagnosis• Collection: • Use Non-toxic cotton swabs (treated with charcoal to absorb toxic fatty acid present in the cotton fiber) • Swabs should be plated immediately (best method) or within 6 hours • Specimen from sterile sites requires no special method in transport like synovial fluids in the syringes, they should be transpotred immediately to the laboratory • Blood culture is an exception, N. gonorrheae and N. meningitidis are sensitive to SPS (Sodium Polyanetholsulfate) which is present in vacutainer tubes, if present should < 0.025% • Transport media: • Amie’s charcoal transport medium • Transgrow medium • New York City medium • JEMBEC
    • Laboratory Diagnosis• G/S & C/S of d/c• Presumptive test – (+) gram-negative intracellular diplococci• Presumptive test – Oxidase test
    • Laboratory Diagnosis• Culture • Gold-standard in diagnosis • Colonies: translucent, grayish, convex, shiny colonies with entire margin, non-hemolytic • TYPES: • T1 & T2  Small, bright reflective colonies, typical of fresh isolates from gonorrheae (+) fimbrae/pili • T3, T4 & T5  Larger, flatter, non-reflecting (-) fimbrae/pili
    • Media Used:• Chocolate agar plate (CAP) • Sterile sites• Thayer-Martin Chocolate (T M) medium • Modified medium of CAP • Non-sterile sites • Vancomycin, Colistin, Nystatin• Modified Thayer-Martin (MTM) • T-M + trimetroprim to (-) swarming Proteus• M-Lewis Agar • Same as T-M but instead of Nystatin, Anisomycin is use
    • Treatment• Ceftriaxone – DOC• Test for Chlamydia trachomatis or treat with tetracycline• Penicillin-binding protein mutations led to gradual increases in penicillin resistance from the 50s to the 70s• Plasmid mediated β lactamase produces high level penicillin resistance
    • PreventionAdult forms: A B C• Abstinence• Be faithful/careful• Condom
    • Prevention• Neonatal: • 0.5 % erythromycin ointment • 1.0 % Tetracycline ointment • 1.0 % Silver nitrate drops (Crede’s prophylaxis)
    • Moraxella catarrhalis• Gram-negative diplococcus (close relative of neisseriae)• Normal upper respiratory flora• Otitis media• Cause bronchitis and bronchopneumonia in elderly with COPD• Drug resistance a problem; most strains produce a β lactamase