MLT 140: Exam 2

Streptococcus Flow Chart

Streptococcus/Enterococcus General Characteristics:

§Colonies:  small, convex, gray and transparent white, can display all three forms of hemolysis depending on  species 

§Gram stain: Gram positive cocci - in chains or pairs, or can be spherical, some elongated, can turn gram negative with age

§Lab ID: catalase (-), hemolytic patterns very important, other tests done when appropriate in order to speciate

§Growth Requirements: Aero-tolerant, facultatively anaerobic organisms, some like increased CO₂

§Normal habitat: throat, nose & nasopharynx, lower GI tract & vagina, EXCEPT for Group A Strep

Streptococcus/Enterococcus Cell Wall Structure

§Thick peptidoglycan layer with techoic acid crosslinks (gram pos.)

§C-carbohydrate layer – C carb used in Lancefield grouping of Strep spp. (Except in S. pnuemo and S. viridans – mostly used for beta streps)

§Capsule – in S. pneumoniae & Group B and in young cultures of most species - prevents phagocytosis

Lancefield Classification: 

§based on C carbohydrate in cell wall

Serological testing and only done on beta-hemolytic streps - Groups A,B,C,D, etc..

§Hemolytic patterns

Alpha – brow/green coloration, partial hemolysis of RBCs in media

Beta – complete lyses the RBCs in the agar, leaves a clear “halo” under and around the colonies

Non-hemolytic (formerly gamma) – no hemolysis

Physiological Characteristics

divides the streptococcal spp. into four groups:

§Pyogenic streptococci - produce pus and they are mostly beta-hemolytic and constitute most of the Lancefield groups

§Lactococci - non-hemolytic with Lancefield group N-antigen and are often found in dairy products

§Enterococci - normal flora in intestines, most lack C-antigen and are not part of the Lancefield classification

§Viridans strep - alpha or non-hemolytic, are normal flora of URT, female genital tract and GI tract, and are opportunistic pathogens

Biochemical Identification

testing based on hemolysis

(1) bacitracin susceptibility (2) CAMP (3) hippurate hydrolysis (4) PYR hydrolysis (5) LAP (6) VP (7)beta-d-glucuronidase (8) bile esculine and salt tolerance (9) optochin susceptibility (10) bile solubility

You have a gpc that is catalase negative. What is the next step in determining additional testing to be done?

Hemolytic pattern

Streptococcus pyogenes, (Group A Strep)

Source:  usually throats, some wounds, urine, blood

Diseases:

§Pharyngitis (Strep Throat)

§Scarlet Fever

§Skin infections:

impetigo - localized skin disease causing weeping lesions

erysipelas - rare skin infection seen in elderly

cellulitis - deeper invasion of the skin seen often in patients with vascular disease or diabetes that can lead to bacteremia, sepsis, or gangrene

§Necrotizing Fasciitis - skin infection, progesses rapidly, fatal, “flesh eating bacteria”

§Streptococcal Toxic Shock Syndrome - organs shut down leads to death, origin unkown, young children and elderly

§Rheumatic Fever - sequalae, damage to heart valves, present after 1 month of infection, rare.

§Acute Glomerulonephritis - sequalae of GAS, inflammatory response to complexes that deposit in the glomerulus causes damage. Leads to impairment in kidney function, more common in children.

§Bacteremia - invasive disease, reportable to local health department

Sequalae

sequel, infection has to occur about a month after the intial GAS infection

Streptococcus pyogenes, (Group A Strep) : Virulence Factors

Capsule – prevents phagocytosis

M protein – resists phagocytosis, adheres to mucous membranes

Lipotechoic acid & Protein F - adhesion molecule, works w/ M protein for attachment

Streptolysin O – tissue invasion & destruction, anaerobic hemolysis, ASO test measures antibodies, immunogenic

Streptolysin S  - tissue invasion/destruction, aerobic hemolysis, non-immunogenic

Streptokinase – lyses fibrin clots

Hyaluronidase – destroys connective tissue, disease spreads

Streptococcal Pyrogenic Exotoxins – aka scarlet fever toxin, super antigens, causes rash seen in scarlet fever

DNase – kill WBCs, antibodies produced, Group A produces 4 different dnases (enzyme)

Streptococcus pyogenes, (Group A Strep): Lab identification

Colony:  small, grayish-white, transparent to translucent, beta hemolytic

Catalase negative

Bacitracin susceptible - aka A disk, streak on a BAP then placing the disk in middle of streaked area, looking for no growth around the A disk - zone can be any size and be considered positive

PYR positive - disk test, pink/red = positive, colorless/no change = negative, enterococcus is also positive for this test so you would have to do more testing to differentiate if the colonies are beta hemolytic, PYR is more specific for GAS than bacitracin.

Bile esculin negative

6.5% NaCl negative

Hippurate hydrolysis negative - positive is purple, negative is colorless, this test is more for detection of Group B

CAMP negative

Treatment: penicillin - GAS is sensitive penicillin and the cephalosporins meaning that sensitivity testing is not necessary because we know how this organism responds to these particular drugs, therefore, when reporting all you really have to do is report the organism and make a statement that the org has predictable sensitivities to those drugs

Bacitracin Sensitivity Test

(A disk). Left side you can see the beta hemolytic zone where the organism was streaked to the plate and the disk was placed in the center of the streak. Hard to see because the zone is small but there is a thin zone around the disk of no growth - any size of no growth is positive (susceptible).

Right side of plate is the same setup though there is no zone around the disk = negative (resistant)

PYR Test

Small paper disk where you apply organism to. After letting the organism set for a couple minutes you then apply a drop of the color developer to see the test reaction. In this case the test is positive meaning the organism could be either Group A Strep or enterococcus - further testing would be necessary to positively ID

Strep agalactiae

“Group B Strep” growing on BAP - small zone of beta hemolysis around colonies. typical of Streps - much smaller zones of hemolysis that is seen in staphs

Streptococcus agalactiae, Group B

Source: vaginal cultures (pregnant women), blood, wounds, urine

Diseases

§Spontaneous abortion – pregnant women screened

§Invasive disease in newborns: most common cause of meningitis in newborns; pneumonia

§Wounds – elderly, immunocompromised

§UTI

Virulence Factors: capsule

Lab identification

§Colony: grayish-white, translucent to opaque, beta hemolytic (small zone)

§Sodium hippurate hydrolysis (+), Bacitracin resistant, Bile esculin (-), 6.5% NaCl (-)

§CAMP test (+) (p.345)

Treatment: penicillin, combo drugs

CAMP Test

Positive control = Group B and negative control = Group A. BAP. S aureus (beta hemolytic) is streaked down the middle. Perpendicular to staph streak, you streak control organisms along with your suspected patient colonies. Group B produces arrowhead of hemolysis that looks like a triangle that is blunted on one point.

Hippurate Hydrolysis Test

Liquid broth test for identification of GBS. The tube on the right is positive for Group B = purple

Strep. pneumoniae

What type of hemolysis?

Streptococcus pneumoniae

General Characteristics

§Inhabits the nasopharyngeal areas of healthy individuals

§Typical opportunist

§Cell wall contains antigen referred to as C Substance – similar to C-carb, reacts w/CRP

Virulence Factors

§Capsular polysaccharide: prevents phagocytosis, no capsule = not pathogenic

§Toxins: hemolysin, IgA protease, neuraminidase, hyaluronidase