streptococcus, enterococcus and other gram-positive cocci

Streptococcus, Enterococcusand other Gram-positive cocci

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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”

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

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

Streptococci and their diseases

S. pyogenes (groupA)

Pharyngitis, scarlet fever, pyoderma, erysipelas, cellulitis,

necrotizingfasciitis, streptococcal toxicshock syndrome, bacteremia, rheumatic fever,glomerulonephritis

Streptococci and their diseases

S. agalactiae(group B)

Neonatalinfections(meningitis, pneumoniae, bacteremia)

Urinary tractinfectionsAmnionitis,EndometritisWound infections

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

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

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

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

Streptococcus pyogenes/physiology & structure

Other cell surface components: componentsof the cell wall

M-like proteinsLipoteichoic acidF protein

Capsule

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

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

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

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

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

Streptococcus pyogenes/Pathogenesis&Immunity

Streptolysin S & OStreptolysine S lyse erythrocytes

leukocytesplateletsresponsible for β-hem.

Streptolysine O lyse erythrocytesleukocytesplateletsantibodies are formedagainst ASO test

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

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

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

Other enzymes

C5a peptidase disrupts C5a (activatephagocyting cells)Hyaluronidase (spreading factor)diphosphopyridine nucleotidase (DPNase): role in pathogenesis is unknown

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)

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

Epidemiology/ Soft-tissue infections

Pyoderma, erysipelas, cellulitisare preceded by skin colonizationafter which the organisms are introduced intothe tissues through a break in the skin

Clinical diseases

Suppurative streptococcal diseaseNonsuppurative streptococcal disease

Suppurative streptococcal disease

PharyngitisPyodermaErysipelasCellulitisNecrotizing fasciitisStreptococcal toxic shock syndromeOther suppurative diseases bakteremia

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

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

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

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%

Nonsuppurative streptococcal disease

Rheumatic feverAcute glomerulonephritis

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

Acute glomerulonephritis

Acute inflammation of the renal glomeruliEdema, hypertension, hematuriaSpecific nephritogenic strains are associatedwith this disease

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

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

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

Treatment

PenisilinErythromycin or oral cephlosporinNewer macrolidesDrainage and surgical debridementRheumatic fever: Longterm antibioticprophylaxis

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)

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

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

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

Diagnosis

Antigen detection: latex agglutinationCulture: enriched mediumBeta-hemolysisNucleic acid-based tests: PCR Identification: CAMP test or hydrolysis of hippurate

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

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

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:

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

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).

Viridans Streptococci: colonization

Colonize the oropharinx, gastrointestinal tract, genitourinary tract

Rarely found on the skin surface, because fattyacids are toxic to them

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

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

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

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

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

S. pneumoniae / diseases

PneumoniaeMeningitisSinusitisOtitis mediaBacteremia

Streptococcus pneumoniae/Pathogenesis&Immunity

The disease manifestations are causedprimarily by the host response to infectionrather than the production of organism-specifictoxic factors

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

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

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

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

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

Epidemiology

A common cause ofbacterial pneumoniaMeningitisSinusitisOtitis mediaBacteremia

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)

Quellung reaction

Polyvalent anticapsularantibodies are mixedwith the bacteria

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

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

Enterococcus

Most frequently isolated & most commonlyresponsible for human disease withstreptococcus among gram-positive cocci16 species in the genusE. faecalis & E. faecium are most commonlyisolated

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

Enterococcus/Pathogenesis&Immunity

Are commensal withlimited potential forcausing diseaseDo not possess toxinsCannot avoid beingengulfed & killed byphagocytic cells

BUT,

CauseSeriousDisease

Enterococcus/Pathogenesis&Immunity

Virulence factorsAdhesive factorsSecrete extracellular proteins(cytolysin)Bacteriocins

Inherently resistant to manyantibiotics

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

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)

Enterococcus/Laboratory diagnosis

Grow readily on nonselective mediaResemble S. pneum.

Differentiation resistant to optochindon’t dissolve whenexposed to bilehydrolyze PYRpyrolidonyl-β-naphthylamide

Other Catalase-negative Gram positivecocci

Leuconostoc (VaR)Pediococcus (VaR)LactococcusAerococcus (air coccus)