MLAB 2434: Microbiology Keri Brophy-Martinez
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Transcript of MLAB 2434: Microbiology Keri Brophy-Martinez
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.