Staphylococcus And Streptococcus

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Gram positive cocci

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High amount of peotidoglycan in cell wall, retain crystal violet, natural habitants of skin and mucous membranes of humans, infections spread through direct contact, and pyogenic infections.

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Color of micrococcus on blood agar

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Yellow.

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Family staphylococcaceae classification

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Staphylococcus, gemella, macrococcus, and salinococcus.

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Characteristics of staphylococcus

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Gram positive in clusters or tetrads, facultative anaerobes, catalase positive, medium creamy colonies in blood and CNA white, yellow, or cream pigments, non motile.

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What plates do you use to isolate staphylococcus?

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CNA or mannitol salt.

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Pyogenic

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Pus forming.

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Coagulase negative staphylococci

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Staph epidermidis and staph saprophyticus.

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Morphology of staphylococci

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Tetrads or clusters.

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Tolerance of salt for staphylococcaceae

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Salt tolerant.

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Purpose of catalase test

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Differentiation of staph from strep.

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First plates to isolate staph and micrococcus.

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CNA, Maconkey, and blood agar.

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CNA

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Selects for gram pos cocci.

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MSA

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Used for isolation of staph because it can sustain high salt. Yellow means fermentation.

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Appearance of staph aureus

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Medium to large colonies. Smooth and creamy appearance. Exhibit beta hemolysis. Some are non hemolytic.

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Two types of coagulase

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Bound and unbound. Bound is detected in the slide coagulase test. Indicates conversion of fibrinogen to fibrin.

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Latex agglutination

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Latex particles are coated with human plasma fibrinogen and immunoglobulin G (IgG), which will react with clumping factor and protein A of staph aureus.

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MSA for staph aureus

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Growth and fermentation.

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DNase results for staph aureus

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Positive.

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DNase positive

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Clearing.

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How does staph aureus cause disease?

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By invading tissues and producing toxins.

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Modified oxidase results

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Micrococcus is positive, staph is negative.

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Catalase results: negative and positive

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Negative: no bubbling.

Positive: bubbling.

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Catalase test

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Hydrogen peroxide added to specimen.

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Content of MSA

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7.5% - 10% NaCl, mannitol, and phenol red indicator.

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Which test is most reliable for differentiation of staph aureus?

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Coagulase.

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Coagulase test and results

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Converts fibrinogen to fibrin clot. Agglutination means positive result. If negative, do coagulase tube test to detect free coag.

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Process of coag test

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Incubate organism with rabbit plasma at 37 degrees and observe for clot.

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Important characteristics of Staph aureus

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Free coag positive, MSA growth and fermentation, and DNase positive.

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What indicates infection?

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Gram positive cocci in clusters with neutrophils.

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Factor function of enterotoxins

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Associated with food-borne diseases or gastroenteritis. Staph aureus virulence factor. Group of 7 heat stable proteins: a, b, c, d, e, h, and I. Most food poisoning associated with A and B.

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Hemolysin

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Exotoxin that lyses RBCs.

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Leukocidan

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Exotoxin that can inhibit white cells.

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Function of protein A

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Binds to fc fraction of most IgG antibody molecules; interferes with opsonization and phagocytosis from neutrophils and activation of complement; affects immediate and delayed hypersensitivity reactions; immunogenic.

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Function of catalase

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Staph aureus virulence factor. Inactivate hydrogen peroxide formed in neutrophils as they invest and destroy bacteria with myeloperoxidase enzymes.

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Function of lipase and what is it associated with?

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Virulence factor of staph aureus. Hydrolyzes lipids in plasma and skin; enables staphylococci to colonize certain body areas; is associated with initiation of skin infections, such as boils, carbuncles, and furnuncles.

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Superantigens function

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Enterotoxins are included in pyrogenic exotoxins. TSST-1 promotes cytosine release and rapid progress of toxic shock syndrome.

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What can methicillin resistant staph aureus be detected with?

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Cefoxitin screen test.

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Vancomycin intermediate staph aureus

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4-8 mcg/mL.

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Vancomycin resistant staph aureus

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Greater than or equal to 16.

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Characteristics of coagulase negative staph

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Catalase positive, GPC clusters, growth on mannitol salt, but no fermentation, DNAse negative, and coagulase negative.

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Identifying characteristics of Staph epidermidis

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GCP in clusters, white, creamy, raised growth in blood agar, no hemolytic on blood agar, positive growth on CNA, growth with no fermentation on MSA, coag negative, DNase negative, and susceptible to novobiocin.

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Staph saprophyticus is considered a ______ unless found in urine. What urinary infection is it associated with?

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Contaminant. UTIs with low colony count.

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Identification staph saprophyticus

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Coagulase negative, DNase negative, grows on MSA with variable fermentation, and resistant to novobiocin.

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Procedure for bound coagulase

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Place drop of rabbit plasma on clean slide. Place drop of sterile saline or distilled water on separate area of the slide. Using sterile inoculating loop, emulsify a suspension of organisms in saline or water. This is the negative control. Emulsify a suspension of the organisms in the plasma. Try to form a smooth suspension. Slide back and forth for 1 minute. Interpret for agglutination.

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Characteristics of streptococcus

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Gram positive cocci 0.5-2.0 in diameter that occur in chains or pairs, nonmotile, nonsporeforming, facultative anerobes, some species show enhanced growth under increased co2, require supportive or enriched agar for growth, and catalase negative.

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Lancefield antigen grouping

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Based on hemolysis.

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Alpha hemolysis

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Incomplete or partial hemolysis; green or brown surrounding the colony.

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Beta hemolysis

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Complete hemolysis of RBCs; clearing or colorless zone around colony.

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Beta hemolysis results from the production of:

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Two hemolysis, streptolysin S and streptolysin O.

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Streptolysin S

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Nonantigenic, oxygen stable, and surface hemolysis.

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Streptolysin O

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Antigenic, oxygen labile, and produced by many group a strep. Use stabs to determine subsurface hemolysis. Inactivated in presence of oxygen.

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Beta hemolytic strep classified based on:

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Specific cell wall carb antigens.

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Diseases associated with strep A

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Bacterial pharyngitis, pyoderma, streptococcal toxic shock like syndrome.

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Group B diseases

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Neonatal bacteremia, pneumonia, and meningitis.

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Group D diseases

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UTI, intra abdominal infections, endocarditis, VRE.

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Group F diseases

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Wound infections.

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Primary infections of strep a

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Bacterial pharyngitis or strep throat. Strep pyogenes.

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What plates do you set a throat culture up on?

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BAP, CNA, MAC, and a chocolate.

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Case study on test

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Gram positive cocci in chains for CSF, white cells.

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Characteristics of group a strep

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Wide zone of beta hemolysis, bacitracin susceptible, pyrase positive, sxt resistant, and CAMP negative.

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Colonial morphology of strep a

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Pinpoint, translucent, clear or opalescent, and wide zone of beta hemolysis.

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Mucous colonies of strep a are caused by:

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M (emm) protein that contain hyaluronic acid.

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Presumptive identification of staph pyogenes is through:

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Bacitracin test and pyr test. Susceptible to bacitracin and pyr positive.

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Streptococcal toxic shock syndrome

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Invasion of soft tissue and necrotizing fasciitis fever, alteration of blood brain barrier.

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Group B strep

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Streptococcus agalactiae.

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Hemolysis for group b strep

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Narrow zone beta hemolytic or non hemolytic.

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Normal flora of group b strep

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May be found in vaginal tract and GI tract.

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Characteristics of group B strep

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Beta hemolytic (narrow) or nonhemolytic, CAMP positive, hippurate positive, bacitracin resistant, SXT resistant, pyrase negative, and bile esculin hydrolysis negative.

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Positive CAMP

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Arrowhead.

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Which strep group should be screened in mothers?

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Group B. Baby may become infected during birth process by inhaling organisms which may lead to pneumonia or meningitis.

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CAMP test

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Synergistic hemolysis between group B streptococcus and Group a. Extra cellular, thermostable, antigenic protein produced by group B strep.

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Hippurate test

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Used to identify group B strep. It hydrolyzes sodium hippurate to benzoic acid and glycine. Benzoic acid is detected with ninhydrin reagent.

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Shape of strep pneumoniae in gram stain

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Lancet or bullet shaped.

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Why is strep pneumoniae isolation plate placed in candle jar?

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Requires CO2.

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Appearance of strep pneumoniae colonies after 24 hours

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Small, round, glistening, dome shaped that are transparent with an entire edge. Mucoid and alpha hemolytic.

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As colonies age, what happens to strep pneumoniae colonies?

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Autolysis. Colonies collapse and appear umbilicated.

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What test identifies strep pneumoniae?

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P-disk. Optochin susceptible.

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P disk procedure

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Streak for lawn of growth. Apply optochin disc. Incubate in co2 for 18-24 hours. Measure zone of inhibition.

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Characteristics of enterococcus

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Isolate on SBA. Bile esculin positive. Grow in 6.5% NaCl. Pyrase negative. Penicillin resistant.

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Bile esculin test

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Presumptive identification of group d antigen.

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What acts as an indicator of esculin hydrolysis? What indicates positive result?

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Ferric citrate. Black = positive.

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What does pyrase differentiate?

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Group D strep and beta hemolytic strep: group a and b.

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VRE subgroups

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Primarily E faecalis and E faecium.