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Streptococcus, Enterococcusand other Gram-positive cocci
Doç Dr Nevriye Gönüllü
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StreptococcusGram positiveGrow pattern pairs, chainsMost species are facultatively anaerobesSome grow only in atmosphere enhancedwith CO2 (capnophilic growth)Nutritional requirements are complex
Blood, serum enriched media“Catalase-negative”
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Classification of common streptococcalpathogens
Biochemical:S.pyogenesS.agalactiaeS.dysgalactiaeS.anginosus groupS.bovisViridans groupS.pneumoniae
Serologic/Hemolysis:Β/βC,G/ βA,C,F,G/βD/ αNongroupable/αNongroupable/α
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StreptococcusClassification is complicated
3 different schemes are used1. Lancefield groupings according to serologic
properties (A-H, K-M, O-V)2. Hemolytic patterns: β, α & γ hemolysis3. Biochemical properties
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Streptococci and their diseases
S. pyogenes (groupA)
Pharyngitis, scarlet fever, pyoderma, erysipelas, cellulitis,
necrotizingfasciitis, streptococcal toxicshock syndrome, bacteremia, rheumatic fever,glomerulonephritis
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Streptococci and their diseases
S. agalactiae(group B)
Neonatalinfections(meningitis, pneumoniae, bacteremia)
Urinary tractinfectionsAmnionitis,EndometritisWound infections
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Streptococcus pyogenes/physiology & structure
Spherical cocciForm short (clinical specimen) or long chains(liquid media)Growth on enriched-blood agar mediaWhite colonies 1-2 mm with large zonesof β-hemolysisEncapsulated strains mucoidBasic structure in the cell wall is peptidoglycan as Gram+ group and typespecific antigens
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Streptococcus pyogenes/physiology & structure
Group specific carbohydrateWithin the cell wall10 % of the dry weightA dimer of N-acetylglucosamine and rhamnoseIs used to classify group A streptococci anddistinguish them from others
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Streptococcus pyogenes/physiology & structure
Type specific proteinsM protein major type-specific protein associated with virulent streptococci
2 polypeptide chains1. Highly conserved among all group A streptococci2. Responsible for the antigenic variability >100
serotypes3. Are subdivided into class I (share exposed
antigens) and class II molecules (do not haveexposed shared antigens)
4. Only bacteria with class I M proteins causerheumatic fever
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Type specific proteins
T protein (trypsin-resistant): useful epidemiologicmarker
Usefull when bacteria fail to express the M proteinStructure unknownEpidemiologic classification based on identificationof specific M and T types by agglutination with M- orT-specific antibodiesWill be replaced by sequencing the emm gene thatencodes the M protein
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Streptococcus pyogenes/physiology & structure
Other cell surface components: componentsof the cell wall
M-like proteinsLipoteichoic acidF protein
Capsule
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Capsule
An outer hyaluronic acid capsule (repeatingmolecules of glucuronic acid and N-acetylglucosamine)Antigenically indistinguishable fromhyaluronic acid in mammalian connectivetissuesFunctions: to prevent phagocytosisEncapsulated strains are responsible forsevere systemic infections
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Streptococcus pyogenes/Pathogenesis&Immunity
Virulence of S. pyogenesThe ability of the bacteria
to adhere to the surface of the host cellsInvade into the epithelial cellsAvoid opsonization & phagocytosisProduce a variety of toxins & enzymes
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Streptococcus pyogenes/Pathogenesis&Immunity
Adherence to host cells is mediate by 10 different bacterial antigensThe most important:
lipoteichoic acidM proteinsF protein
Invasion into epithelial cells is mediate by M protein and F protein
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Streptococcus pyogenes/Pathogenesis&Immunity
Pyrogenic exotoxins(Streptococcal pyrogenic exotoxin, Spes), originally called erythrogenic
toxins
Produced by lysogenic strains, similar to thetoxin produced in Corynebacterium diphteriaeFour immunologically distinct heat-labile toxins(SpeA, SpeB, SpeC and SpeF) Superantigens (IL-1, IL-2, IL-6, TNF-β/α
Responsible for the streptococcal toxic shocksyndrome
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Streptococcal pyrogenic exotoxin, (Spes)
Act as superantigens, interacting withmacrophages and helper T cellsSecreted cytokines mediate important effects, including shock and the organ failure seen in patients with streptococcal toxic shocksyndromeResponsible for the rash observed in scarletfever
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Streptococcus pyogenes/Pathogenesis&Immunity
Streptolysin S & OStreptolysine S lyse erythrocytes
leukocytesplateletsresponsible for β-hem.
Streptolysine O lyse erythrocytesleukocytesplateletsantibodies are formedagainst ASO test
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Streptolysin S & O
Streptolysin S:Oxygen-stableNonimmunogenicProduced in thepresence of serum
Streptolysin O:Oxygen-labileAnti-ASO test useful fordocumenting recentgroup A streptococcalinfectionAntigenically related totoxins producedby:S.pneumoniae, C.tetani, C.perfringens
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Streptococcus pyogenes/Pathogenesis&Immunity
StreptokinasesTwo forms: A & BMediate the cleavage of plasminogen, releasingthe protease plasmin thatCleaves fibrin and fibrinogen, resulting in the lysisof clots and fibrin depositsLyse blood clots, fibrin deposits, facilitate the rapidspread of bacteriaAnti-streptokinase antibodies: useful marker forinfection
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Streptococcus pyogenes/Pathogenesis&Immunity
DeoxyribonucleasesFour immunologically distinct: DNAases A to DNot cytolyticDepolymerase free DNA in pus: reduce viscosityof the abscess, facilitate spreadDeveloped antibodies: important markers of cutaneous infections
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Other enzymes
C5a peptidase disrupts C5a (activatephagocyting cells)Hyaluronidase (spreading factor)diphosphopyridine nucleotidase (DPNase): role in pathogenesis is unknown
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Epidemiology
Colonize the oropharynx of healthy childrenand young adultsCarriage 15-20%Other streptococcal species (S.anginosus, S.intermedius, S.constelatus) can carrygroup-specific A antigen: do not causepharyngitisIs transient (bacteriocins produced by otherstreptococci suppress the growth)
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Epidemiology
Disease caused by recently acquired strainsPrimarily infect children between 5-15 yearsSpread from person to person throughrespiratory dropletsCrowding, classrooms, daycare facilitiesParticularly during the winter months
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Epidemiology/ Soft-tissue infections
Pyoderma, erysipelas, cellulitisare preceded by skin colonizationafter which the organisms are introduced intothe tissues through a break in the skin
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Clinical diseases
Suppurative streptococcal diseaseNonsuppurative streptococcal disease
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Suppurative streptococcal disease
PharyngitisPyodermaErysipelasCellulitisNecrotizing fasciitisStreptococcal toxic shock syndromeOther suppurative diseases bakteremia
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Pharyngitis Scarlet fever
Develops 2 to 4 daysafter exposure to thepathogenSore throut, fever, malaise, and headachePosterior pharynxerythematousWith exudateCervicallymphadenopathy
Complication of pharyngitisStrain lysogenized by a temperatebacteriophageStimulates productionof a pyrogenic exotoxinRash, Pastia’linesStrawberry tongue
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Pyoderma Erysipelas
Purulent (“pyo“) infection of the skin (“derma“)Infects exposed areas(face, legs)Introduced into thesubcutaneous tissueThrough a break in theskin
Erythros “red“; pella“skin“Involved skin area is typically raisedYoung children andolder adults
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Cellulitis Necrotizing fasciitis
Involves the skin anddeeper subcutaneoustissuesDistinction of infectedskin is not as clearLocal and systemicsigns
Also calledstreptococcal gangreneOccurs deep in thetissueExtensive destruction of muscle and fat“flesh-eating bacteria“Gangrene and systemicsymptoms
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Strept.toxic shock syndrome Other
M serotypes 1 or 3Prominent hyaluronicacid capsulesPyrogenic exotoxins: SpeA and SpeCMultisystem toxicityShock and organ failure(kidney, lungs, liver)
Puerperal sepsisPneumoniaBacteremia in patientswith necrotizing fasciitisor toxic shocksyndromeMortality 40%
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Nonsuppurative streptococcal disease
Rheumatic feverAcute glomerulonephritis
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Rheumatic fever
NonsuppurativecomplicationInvolvement of the heartvalvesMigratory arthritisAssociated withstreptococcal pharyngitisIf antibiotic prophylaxis is not used
Diagnosis is made on thebasis of clinical findings andfindings of a recentS.pyogenes infection:
culture resultsDetection of group A AgElevation of ASO, anti-DNase B, anti-hyaluronidase antibodies
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Acute glomerulonephritis
Acute inflammation of the renal glomeruliEdema, hypertension, hematuriaSpecific nephritogenic strains are associatedwith this disease
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Streptococcus pyogenes/IDENTIFICATION
Microscopy: gram +cocci in pairs and chainsAntigen detection: use antibodies that reactwith the group-specific carbohydrate in thebacterial wallEnzyme immunassay (EIA) or bound to latexparticlesSensitivity is low (90%): all negative resultsmust be confirmed by culture
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Streptococcus pyogenes/IDENTIFICATION
Culture: specimens must be obtained from theposterior oropharynx or skin lesionsNutritionally enriched agar media with sheepblood
Selective media (media with “bactrim”)Prolonged incubation
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Identification
Susceptibility to bacitracinL-pyrrolidonyl arylamidase (PYR): hydrolyzesL-pyrrolidonyl-β-naphtylamide releasing β-naphthylamideAntibody detection:antibodies againststreptolysin O (the ASO test); appear 3-4 weeks after the exposureAnti-DNase B test if glomerulonephritis is suspected
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Treatment
PenisilinErythromycin or oral cephlosporinNewer macrolidesDrainage and surgical debridementRheumatic fever: Longterm antibioticprophylaxis
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Streptococcus agalactiae (Group B)The only species that carries the group B antigenphysiology & structure
Gr + cocciShort or long chains (indistinguishable from S. pyogenes)
Buttery colonies, narrow zone of β-hemolysisSubdividing
The B antigen group specificCapsular polysaccharides type-specificC protein (surface protein)
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Streptococcus agalactiae/IDENTIFICATION
MicroscopyCulture:
Readily grow on a nutritionally enriched mediumLarge coloniesβ-hemolysis may be absent selective brothmedium with antibiotics
IdentificationPreliminary identification (+) CAMP test,
hydrolysis of hippurate
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Pathogenesis
Genital colonization with group B streptococcihas been associated with increased risk of premature deliverySerotypes Ia, III and V are most commonlyassociated with colonization and diseaseColonize lower gastrointestinal tract andgenitourinary tract60% of infants born become colonized withtheir mothers’ organisms
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Early/Late –onset disease
Septicemia and meningitis in newbornsEarly: infants younger than 7 daysLate: 1 week-3 months
In adults:urinary tract infections, woundinfections, skin and soft-tissue infections
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Diagnosis
Antigen detection: latex agglutinationCulture: enriched mediumBeta-hemolysisNucleic acid-based tests: PCR Identification: CAMP test or hydrolysis of hippurate
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Treatment
Penisilin: needs greater MICTolerance to penicilin has beem reportedCombination of penicillin and aminoglycosidein serious infections or vancomycin in penicillin allergyPrevention: all pregnant women should be screened for colonization at 35-37 weeks of gestation
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Other beta-hemolytic streptococciGroup C, F and G are most commonlyassociated with human disease2 species of particular importance
S. anginosusS. dysgalactiaePosses group A, C, F or G capsularpolysaccarideAbscess formation, bacteremia
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Viridans Streptococci
A heterogeneous colection of α-hemolytic andnonhemolytic streptococciTheir name is derived from viridis (Latin forgreen)Produce a green pigment on blood agarmediaMore than 20 species; are classified into fivesubgroups:
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Viridans Streptococci: subgroups
Anginosus: S.anginosus, S.intermediusMitis: S.mitis, S.pneumoniae, S.sanguisMutans:S.mutans, S.sobrinusSalivarius: S.salivarius, S.vestibularisBovis:S.bovis, S.equinusUngrouped: S.suis
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Viridans Streptococci: culture
Require complex media supplemented withblood products and an incubation atmosphereat 5-10 % CO2.Some strains are “nutritionally deficientstreptococci“ :Can grow only in the presence of exogenouslysupplied pyridoxal (the active form of vitamin B6)These organisms can grow in blood cultures, but cannot grow when subcultured (unless pyridoxalsupplemented media are used).
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Viridans Streptococci: colonization
Colonize the oropharinx, gastrointestinal tract, genitourinary tract
Rarely found on the skin surface, because fattyacids are toxic to them
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Viridans Streptococci: infections
Associated with dental caries, subacuteendocarditis, and suppurative intraabdominalinfections
Subacute bacterial endocarditis is associatedwith S.gordonii, S.mitis, S.mutans, S.oralis, S.sanguis
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Antibiotic resistance
In the past they were highly susceptible topenicillin (MIC <0.1µg/ml)Moderately and highly resistant strains havebecome commonTreatment:
Penicillin+aminoglycosideBroad spectrum cephalosporin+vancomycin
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Streptococcus pneumoniae
Isolated independently by Pasteur andSteinberg more than 100 years agoIs still a leading cause of morbidity andmortalityEncapsulated, Gr + coccusOval or lancet-shaped cells, in pairs or shortchainsα-hemolytic: production of pneumolysin , an enzyme that degrades hemoglobin producing a green product
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Streptococcus pneumoniae
Can grow only on enriched media (with bloodproducts)Catalase (-)Polysaccharide capsule is used for theserologic classification:
90 serotypesCapsular polysaccharides are used in vaccines
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Streptococcus pneumoniae: cell wall
Peptidoglycan layer is typical of gram-positivecocciTeichoic acid, rich in galactosamine and cholineC polysaccharide (CRP): precipitates a serum globulin fraction (C-reactive protein)F antigen: lipid-bound teichoic acid in thebacterial cytoplasmic membrane
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S. pneumoniae / diseases
PneumoniaeMeningitisSinusitisOtitis mediaBacteremia
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Streptococcus pneumoniae/Pathogenesis&Immunity
The disease manifestations are causedprimarily by the host response to infectionrather than the production of organism-specifictoxic factors
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Streptococcus pneumoniae/Pathogenesis&Immunity
Colonization and migrationS.pn colonizes the oropharynxCan spread to the lungs, paranasalsinuses, middle ear, blood stream
By means of:Surface protein adhesinssecretory IgA (sIgA) proteasePneumolysin: a cytotoxin similar to thestreptolysin O in S.pyogenes
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Streptococcus pneumoniae/Pathogenesis&Immunity
Tissue destructionMobilization of inflamatory cells characteristic of pneumococcal infections
Teichoic acid and peptidoglycan fragments: activate alternative complement pathway producingC5aPneumolysin: activates the classic complementpathway, resulting in prodution of C3a and C5a H2O2 production: lead to tissue damagePhosphorylcholin: binds phosphodiesterase-activating factor, allowing bacteria to enter hostcells
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Streptococcus pneumoniae/Pathogenesis&Immunity
Phagocytic survivalSurvive phagocytosis because of the antiphagocyticprotection afforded by its capsulePneumolysin mediates suppression of thephagocytosisThe virulence is a direct result of this capsule
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Epidemiology
Common inhabitant of the throat andnasopharynx in healthy peapleA 5-75% incidence of such carriage has beenreportedColonization more common in children thanin adultsOccurs at approximately 6 months of age
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Epidemiology
Pneumococcal disease occurs whenorganisms colonizing the nasopharynx andoropharynx spread to distal locci:The lungs (pneumonia)Paranasal sinuses (sinusitis)Ears (otitis media)Meninges (meningitis)Bacteremia
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Epidemiology
A common cause ofbacterial pneumoniaMeningitisSinusitisOtitis mediaBacteremia
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Streptococcus pneumoniae/IDENTIFICATION
MicroscopyLancet-shaped, Gr (+) diplococci, unstainedcapsule (Gram stain with “quellung” reaction)
Culture:Enriched supplemented medium with bloodSelective medium with gentamicin
IdentificationBile solubility testOptochin (ethylhydrocupreine dihydrochloride)
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Quellung reaction
Polyvalent anticapsularantibodies are mixedwith the bacteria
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Streptococcus pneumoniae-Antibiotic resistance
Resistance to multiple antibiotics, includingpenicillin was reportedMechanism: Decreased affinity of theantibiotic for the penicillin-binding proteinspresent in the bacterial cell wall
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Enterococcus
The enterococci (“enteric cocci”) werepreviously classified as group D streptococciThey possess the group D cell wall antigen(glycerol teichoic acid)Are distinct from other group D streptococci(nonenterococcal group D streptococci e.g., Streptococcus bovis)1984: the new genus Enterococcus
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Enterococcus
Most frequently isolated & most commonlyresponsible for human disease withstreptococcus among gram-positive cocci16 species in the genusE. faecalis & E. faecium are most commonlyisolated
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Enterococcus/physiology & structureThey can not be differentiated from S. pneumoniae in microscopeFacultatively anaerobicOptimal growth temp.= 35 oC (10oC to 45oC)White, large colonies on blood agar (after 24h)
Nonhemolytic (or α or β-hemolysis)Grow in the presence of
6.5 % NaCl, tolerate 40 % bile salts, hydrolyse esculin
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Enterococcus/Pathogenesis&Immunity
Are commensal withlimited potential forcausing diseaseDo not possess toxinsCannot avoid beingengulfed & killed byphagocytic cells
BUT,
CauseSeriousDisease
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Enterococcus/Pathogenesis&Immunity
Virulence factorsAdhesive factorsSecrete extracellular proteins(cytolysin)Bacteriocins
Inherently resistant to manyantibiotics
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Epidemiology
Are enteric bacteriaRecovered in feces collected from humansand animalsE.g., E.faecalis 107 per gram fecesE. faecium are less frequentlyE.gallinarum and casseliflavus: colonize theintestine in small numbers; resistant tovancomycin; can be confused with the moreimportant species
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Enterococcus/Clinical DiseasesCan cause life-threatening infectionsOne of the most feared nosocomialpathogens 10% of all nos. infct.Most commonly involved sites
Urinary tractBlood stream
A sever complication: endocarditis( followingbacteremia)
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Enterococcus/Laboratory diagnosis
Grow readily on nonselective mediaResemble S. pneum.
Differentiation resistant to optochindon’t dissolve whenexposed to bilehydrolyze PYRpyrolidonyl-β-naphthylamide
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Other Catalase-negative Gram positivecocci
Leuconostoc (VaR)Pediococcus (VaR)LactococcusAerococcus (air coccus)