MLAB 2434: MicrobiologyMLAB 2434: MicrobiologyKeri Brophy-MartinezKeri Brophy-Martinez

Streptococci, Enterococci and Other Catalase-Negative Gram Positive Cocci

StreptococcusStreptococcus and and EnterococcusEnterococcus: General : General CharacteristicsCharacteristics

Members of the Streptococcaceae family

Facultatively anaerobic

Aerotolerant

Catalase negative

StreptococcusStreptococcus and and EnterococcusEnterococcus: General : General CharacteristicsCharacteristics

Most are typically spherical; some may appear elongated or ovoid

They may appear in chains or pairs

StreptococcusStreptococcus and and EnterococcusEnterococcus: :

Habitat and Clinical Infections Habitat and Clinical Infections

Habitat◦ Normal Flora

Respiratory tract Gastrointestinal

tract Urogenital tracts

Clinical Infections◦ Upper and lower

respiratory tract infections

◦ Urinary tract infections

◦ Wound infections

◦ Endocarditis

StreptococcusStreptococcus and and EnterococcusEnterococcus::Cell Wall StructureCell Wall Structure

Thick peptidoglycan layer

Teichoic acid

Carbohydrate layer present ◦ Used in Lancefield grouping of

Streptococcus spp.

Capsule◦ Virulence factor

◦ S. pneumoniae

Classification OverviewClassification Overview Physiologic characteristics

◦ Pyogenic: produce pus◦ Lactococci: found in dairy products◦ Enterococci: normal gut flora◦ Viridans: normal URT flora

Hemolysis◦ J. H Brown

◦ Alpha, beta, gamma classifications

Serological grouping◦ Typing of C carbohydrate

◦ Lancefield group

◦ Performed only on β-hemolytic hemolysis

Biochemical◦ Based on reaction of isolate

Classification:Classification:HemolysisHemolysis

J.H. Brown- 1903Grouped streps on ability to lyse

RBCS◦Alpha◦Beta◦Gamma◦Alpha-prime

Hemolysis PatternsHemolysis Patterns

◦Alpha (α): Greenish discoloration

Caused by partial lysis of RBCs in media

Hemolysis PatternsHemolysis Patterns

Beta (ß):◦ Complete lysis of

RBCs◦ Produces a clear,

colorless zone

Hemolysis PatternsHemolysis Patterns

◦Gamma : Colonies show no hemolysis or discoloration

Called non-hemolytic

Classification:Classification:Serological GroupingSerological Grouping

Rebecca Lancefield – 1930Based on presence of

carbohydrates in cell wallGroups A, B, C, and D most

significantTyping done on beta-hemolytic

colonies

Classification:Classification:Biochemical Biochemical Identification/SusceptibilityIdentification/Susceptibility

Bacitracin ◦ “A” disk or “Taxo

A” disk◦ 0.04 units◦ Identifies Group A

streptococci (S. pyogenes)

◦ Zone of inhibition is presumptive ID of Grp. A strep

Group A streptococcus is susceptible to “A” disk (left)

Biochemical Biochemical Identification/SusceptibilityIdentification/Susceptibility

Optochin ◦ P disk or“Taxo P” disk◦Differentiates S. pneumoniae from other alpha-hemolytic streptococci

Biochemical IdentificationBiochemical IdentificationBile solubility test

◦Detects amidase enzyme◦Under bile salt or detergent lyses cell

wall Clearing through lysis of colonies

◦Diagnostic for S. pneumoniae

Biochemical IdentificationBiochemical Identification

PYR hydrolysis◦ Substrate L-pyrrolidonyl-

napthlyamide (PYR) is hydrolyzed by the enzyme pyrrolidonyl arlamide

◦ Group A Streptococci and Enterococcus sp. posses the necessary enzyme.

◦ More specific than Bacitracin for Group A streptococci

The disk on the right has turned a red color, indicating a indicating a positive reaction. The left disk remains a yellow color indicating a negative result.

Biochemical Biochemical IdentificationIdentificationHippurate hydrolysis

◦Differentiates Group B streptococci from other beta hemolytic streptococci

◦Group B streptococci hydrolyzes sodium hippurate forming a purple color

Biochemical IdentificationBiochemical Identification

CAMP test◦ Christie,Atkins, Munch-

Petersen ◦ Detects the production of

enhanced hemolysis that occurs when -lysin and the hemolysins of Group B streptococci come in contact with each other

Group B streptococci showing the classical “arrow-shaped hemolysis near the staphylococcus streak

Biochemical IdentificationBiochemical IdentificationBile esculin

hydrolysis◦Ability to grow in

bile and hydrolyze Esculin

◦Characteristic of streptococci that possess group D antigen and Enterococci

Both Group D streptococci and enterococci produce a positive (top) bile Esculin hydrolysis test.

Biochemical Biochemical IdentificationIdentificationSalt Tolerance

◦ Growth in 6.5% NaCl broth

◦ Differentiates Group D streptococci from enterococci

◦ Enterococcus= POSITIVE Tube on left

◦ Group D Streptococcus= NEGATIVE Tube on right

Non-culture IdentificationNon-culture IdentificationSlide agglutination kits

◦ Latex beads are coated with group specific anti-serum, which clump when mixed with a small amount of colony from the specific Streptococcus sp.

Nucleic Acid Probes◦Detect genes for specific groups

Slide Agglutination TestsSlide Agglutination Tests

Slide Agglutination TestsSlide Agglutination Tests

Break Time!Break Time!

Virulence Factors:Virulence Factors: Streptococcus pyogenes Streptococcus pyogenes

Fimbrae: Protein F◦ Attachment and adherence

M protein:◦ Resistance to phagocytosis

Hyaluronic acid capsule: ◦ Prevents phagocytosis

Lipoteichoic acid: ◦ Adheres to molecules on

host epithelial cells

Virulence Factors:Virulence Factors:Streptococcus pyogenesStreptococcus pyogenes

Hemolysins◦ Streptolysin O (O2 labile) detected in ASO titers

◦ Streptolysin S (O2 stable) Causes hemolysis on plates

Erythrogenic toxin/Streptococcal pyogenic exotoxin:◦ Scarlet fever

Enzymes◦ Streptokinase◦ DNases◦ Hyaluronidase – “spreading factor”

Clinical Conditions:Clinical Conditions: Streptococcus pyogenes Streptococcus pyogenes(Group A) (Group A)

Clinical Conditions:Clinical Conditions: Streptococcus pyogenes Streptococcus pyogenes(Group (Group A) A)

Pyodermal infections◦ Impetigo: weeping

lesion◦ Erysipelas

Cellulitis

Wound InfectionsErysipelas due to Streptococcus pyogenes

Clinical Conditions:Clinical Conditions: Streptococcus pyogenes Streptococcus pyogenes(Group A) (Group A)

Scarlet Fever◦Starts with pharyngitis and causes rash

on trunk and extremities◦Due to untreated Group A infections

Invasive Group A Invasive Group A Streptococcal InfectionsStreptococcal Infections

Streptococcal toxic shock syndrome◦ Multi-organ system failure similar to

staphylococcal toxic shock◦ Initial infection may have been pharyngitis,

cellulitis, peritonitis, or other wound infections

Invasive Group A Invasive Group A Streptococcal InfectionsStreptococcal InfectionsCellulitis/Necrotizing Fasciitis

◦ Severe form of infection that is life-threatening◦ Bacteremia and sepsis may occur◦ In patients necrotizing fasciitis, edema,

erythema, and pain in the affected area may develop

◦ Streptococcal myositis resembles clostridial gangrene

Post–Group-A Post–Group-A Streptococcal InfectionsStreptococcal InfectionsRheumatic fever

◦ Fever◦ Inflammation of the heart, joints, blood

vessels, and subcutaneous tissues◦ Chronic, progressive damage to the heart

valves (most evidence favors cross-reactivity between Strep. antigens and heart tissue)

◦ ASO titer will be elevated

Post–Group-A Post–Group-A Streptococcal InfectionsStreptococcal Infections

Acute glomerulonephritis (AGN)◦ Follows either cutaneous or pharyngeal

infections◦ More common in children than adults◦ Antigen-antibody complexes deposit in the

glomerulus◦ Inflammatory response causes damage to

the glomerulus and impairs the kidneys

Laboratory Diagnosis: Laboratory Diagnosis: Group A StreptococcusGroup A Streptococcus

Grams stained wound smear showing gram-positive cocci in chains with numerous “polys” (PMNs)

Laboratory Diagnosis: Laboratory Diagnosis: Group A StreptococcusGroup A Streptococcus

Colony morphology◦ Transparent, smooth,

and well-defined zone of complete or- hemolysis

Laboratory Diagnosis: Laboratory Diagnosis: Group A StreptococcusGroup A Streptococcus

Identification◦ Catalase-negative◦ Bacitracin-

susceptible◦ PYR-positive◦ Hippurate

hydrolysis- negative◦ Slide agglutination

Group A streptococci is susceptible to Bacitracin disk (left); The right shows resistance

Group B Group B -Hemolytic -Hemolytic Streptococcus (Streptococcus (Streptococcus Streptococcus agalactiaeagalactiae))Colonize the urogenital tract of pregnant

women (10-30% rate – can cause OB complications such as premature rupture of membranes and premature delivery)

Mother fails to pass protective antibodies to fetus

Cause invasive diseases in newborns◦ Early-onset infection◦ Late-onset disease

Invasive Disease in the Invasive Disease in the NewbornNewborn

Early Onset Late-Onset

Age of Onset < 7 days 7 – 30 days

Median age of onset 1 hour 27 days

Maternal complications of labor

Common Less common

Incidence of prematurity 25% Less common

Source of Organism Maternal genital tract Maternal genital tract; nosocomial; community

Clinical presentation Nonspecific (35-55 %)Meningitis 5-10 %Respiratory diseases 35-55 %

FocalMeningitis 25-35 %

Types I, II III, V III (75%)

Mortality Rate 5-15 % 2-10 %

InvasiveInvasive Streptococcus Streptococcus agalactiaeagalactiae Infections Infections

In adults◦ Occurs in immunosuppressed patients or

those with underlying diseases◦ Often found in a previously healthy adult

who just experienced childbirth

Laboratory Diagnosis: Laboratory Diagnosis: Streptococcus agalactiaeStreptococcus agalactiae

Colony morphology◦ Small◦ Grayish-white◦ Mucoid, creamy◦ Narrow zone of -

hemolysis

Laboratory Diagnosis: Laboratory Diagnosis: Streptococcus agalactiaeStreptococcus agalactiae

Presumptive Identification tests◦ Gram stain- GPC in

chains◦ Catalase-negative◦ Bacitracin-resistant◦ Bile esculin- negative◦ Does not grow well in

6.5% NaCl.◦ CAMP- positive◦ Slide agglutination

S. agalactiae shows the arrow-shaped hemolysis near the staphylococcus streak, showing a positive test for CAMP factor

Streptococcus Streptococcus pneumoniaepneumoniae

General characteristics◦ Inhabits the nasopharyngeal areas of healthy

individuals◦ Typical opportunist◦ Possess C substance

Virulence factors◦ Polysaccharide capsule

Clinical Conditions:Clinical Conditions:Streptococcus pneumoniaeStreptococcus pneumoniae

Pneumonia ◦ Most common cause of bacterial pneumonia

Meningitis

Bacteremia

Sinusitis/otitis media ◦ Most common cause of otitis media in children < 3

years

Laboratory Diagnosis:Laboratory Diagnosis:Streptococcus pneumoniaeStreptococcus pneumoniae

Microscopic morphology◦ Gram-positive cocci

in pairs; lancet-shaped (somewhat oval in shape)

Laboratory Diagnosis:Laboratory Diagnosis:Streptococcus pneumoniaeStreptococcus pneumoniae

Colony morphology◦ Smooth,

glistening, wet-looking, mucoid

◦ -Hemolytic◦ CO2enhances

growth◦ As colony ages,

autolytic collapse causes “checker shape”

Laboratory Diagnosis: Laboratory Diagnosis: Streptococcus pneumoniaeStreptococcus pneumoniae

Identification◦ Catalase negative◦ Optochin-

susceptibility-test–susceptible

◦ Bile-solubility-test–positive

Identification SchemaIdentification Schema

EnterEnterococcus ococcus Species Species

Clinically Significant Isolates◦ E. faecalis◦ E. faecium

Opportunistic pathogens◦ In the GI tract, genitourinary tract and oral cavity

Associated infections◦ Bacteremia◦ Urinary tract infections◦ Wound infections◦ Endocarditis◦ Hospital-acquired Infections

Laboratory Diagnosis: Laboratory Diagnosis: Enterococcus Enterococcus SpeciesSpecies

Microscopic morphology◦ Cells tend to elongate

Colony morphology◦ Small, grey◦ Most are non-

hemolytic, although some may show or, rarelyhemolysis

◦ Possess Group D antigen

Laboratory DiagnosisLaboratory Diagnosis:: Enterococcus Enterococcus SpeciesSpecies

Identification tests◦ Catalase: may produce a weak catalase reaction

◦ Hydrolyze bile esculin

◦ Differentiate Group D from Enterococcus sp. with 6.5% NaCl or PYR test

◦ Important to identify Enterococcus from non-Enterococcus, because Enterococcus must be treated more aggressively.

Identification SchemaIdentification Schema

Or PYR disk

Other Streptococcal Other Streptococcal SpeciesSpecies

Viridans group (Viridans means “green”)◦ Members of the normal oral, nasopharyngeal flora, GI

tract and female genital tract◦ Most are hemolytic but also includes nonhemolytic

species◦ The most common cause of subacute bacterial

endocarditis (SBE)

◦ Also involved with gingivitis and dental carries

◦ PYR= negative◦ Optochin= negative◦ Bile solubility= negative

ViridansViridans

5 groups◦ Anginosus

S. anginosus, S. intermedius, S. constellatus◦ Mitis

S. sanguig, S. parasanguis, S. gordonii, S. crista, S. infantis, S. mitis, S. oralis, S. oralis, S. peroris

◦ Mutans S. criceti, S. downei, S. macacae, S. mutans, S.

rattus, S. sobrinus◦ Salivarius

S. salivarius, S. thermophilus, S. vestibularis◦ Bovis

S. equinus, S. gallolyticus,S. infantarius, S. alactolyticus

AbiotrophiaAbiotrophia & & GranulicatellaGranulicatella

◦Once referred to as Nutritionally variant streptococci (NVS)

◦Causes endocarditis and otitis media

◦Normal flora of oral cavity◦Requires pyridoxal to grow (can

satellite around Staph, E. coli, Klebsiella, Enterobacter and yeasts)

StreptococcusStreptococcus and and EnterococcusEnterococcus

Species Hemolysis Group Antigen

Common Terms

Disease Association(s)

S.pyogenes ß A Group A streptococci

Pharyngitis; scarlet fever pyoderma; rheumatic fever; AGN

S.agalactiae ß B Group B streptococci

Neonatal sepsis; puerperal fever; pyogenic infections; pneumonia; meningitis

S. equisimilis ß C Group C streptococci

Pharyngitis; impetigo; pyogenic infections

E. faecalis E. faecium E. durans S. bovis S. equinus

Alpha or no hemolysis ( rarely ß ) Alpha ()or none (rarely ß)

D

D

Enterococci

Nonenterococci

Urinary tract infections Wound infections Bacteremia; Endocarditis Urinary tract; pyogenic infections; Endocarditis infections

S. pneumoniae Alpha () hemolysis

Pneumococcus Bacteremia; pneumonia; meningitis;

Viridans and Nonhemolytic S. sanguis S. salivarius S. mitis or nonhemolytic S. milleri S. mutans Other species

Alpha () hemolysis or

no hemolysis

- Viridans strep Endocarditis Dental caries

StreptococcusStreptococcus and and EnterococcusEnterococcusTreatment

◦Generally, streps are not routinely tested for susceptibility since penicillin drug of choice. If the patient is allergic to pen use erythromycin.

◦Antibiotic resistance seen with Enterococcus, use vancomycin

ReferencesReferenceshttp://archive.microbelibrary.org/ASM

Only/Details.asp?ID=2566 http://www.goodtoknow.co.uk/health/S

carlet-feverhttp://onwardstate.com/2009/12/10/ke

ep-your-goals-to-yourself/Mahon, C. R., Lehman, D. C., &

Manuselis, G. (2011). Textbook of Diagnostic Microbiology (4th ed.). Maryland Heights, MO: Saunders.