1

Gram Positive Bacteria

Dental Optometry MicrobiologyKen B. Waites, M.D., F(AAM)

September, 2008

Objectives• To review and discuss:

• microbiological characteristics• epidemiology• virulence factors• associated diseases• laboratory detection

of clinically important gram-positive cocci and gram-positive bacilli.

• Organisms to be discussed include:– Staphylococcus– Streptococcus– Enterococcus– Listeria– Corynebacterium– Bacillus

2

Staphylococcus• Most important

genus in FamilyMicrococcaceae

• Other genera– Stomatococcus– Micrococcus

Habitat• S. aureus

– anterior nares 50-75% healthy people– skin & mucous membranes– hospital environment

• S. epidermidis & others– resident skin flora, gut, upper

respiratory tract• S. saprophyticus

– Urinary tract in young women

3

Lab Characteristics

• Gram-positive cocci(1 µm) in clusters

• Culture - Aerobic• S. aureus is β

hemolytic• Coagulase• Catalase• Mannitol

fermentation

S. aureus

blood agar mannitol salt agar

Catalase Test for DistinguishingStaphylococci from Streptococci

H2O2 catalase O2 + H2O

Bubbles

Strep. Negative Staph. positive

4

Tube Coagulase Test

• Free coagulasesecreted by S. aureusbut not CNS

• Clots rabbit plasma

Cell Wall of S. aureus

5

Antigenic Structures & VirulenceFactors of S. aureus

• Cell wall peptidoglycan– elicits production of IL-1 and opsonic antibody– PMN chemotaxis “pyogenic”– induces sepsis– activates complement– teichoic acid binds fibronectin on host cells

• Protein A - binds Fc of IgG• Capsule (some strains) antiphagocytic

S. aureus Soluble Virulence Factors

• Catalase - reduce phagocyte killing - remove H2O2

• Coagulase - clots plasma (free & bound)• Hyaluronidase - destroys connective tissue• Beta lactamase - destroys beta lactam drugs• Altered Penicillin binding proteins (PBP2’)• Fibrinolysin• Lipases• Nucleases

6

S. aureus Soluble Virulence Factors• Cytotoxins & leukocidins

– lyse white blood cells (Panton-Valentine)– release lysosomal enzymes → damage tissue

• Exfoliatin– interrupts intercellular skin junctions– “Scalded Skin Syndrome”

• Toxic Shock Toxin – stimulates T cells → cytokines,– endothelial damage → rash– “Toxic Shock Syndrome”

• Enterotoxins– stimulate vomiting by interaction with GI neural

receptors (food poisoning)

S. aureus DiseasesSkin and soft tissue

infectionsFurunclesCarbunclesWound infectionsCellulitisImpetigoBacteremia

EndocarditisCNS Infections

Brain abscess Meningitis - rare Epidural abscess

Impetigo

7

S. aureus DiseasesPulmonary Infections

embolicaspiration

Musculoskeletalosteomyelitisseptic arthritis

Genitourinary Tractrenal carbunclelower UTI

Toxin mediated diseases• scalded skin syndrome• food poisoning• toxic shock disease

Toxic shock

Scalded skin

Treatment of Staph. Infections

• Increase in oxacillin-resistant organisms thatare resistant to most other antibiotics exceptvancomycin (MRSA)

• Nosocomial infections• Recent emergence of virulent community-

acquired MRSA in skin & soft tissue infection• First high-level vancomycin-resistant S.

aureus reported July 2002 contains vanAgene from Enterococcus

8

Coagulase negativestaphylococci

• Opportunistic infections– bacteremias– endocarditis– neutropenic patients– Neonates– UTIs (S. saprophyticus)

• Polysaccharide “slime”– protects bacteria– mediates attachment to catheters, etc.

Staphylococcus epidermidisStaphylococcus epidermidis on sheep blood agar on sheep blood agar

White non-hemolytic colonies

Streptococcaceae

• Streptococcus• Enterococcus

Gram stain of staph (clusters)and strep (chains)

Staph

Strep

9

Streptococcus Classification• Hemolysis

– beta– alpha– gamma

• Lancefield Groups– (A-T- β hemolytic)– group-specific cell wall

polysaccharide• Species

– phenotypic biochemicalreactions

Hemolytic Reactions

β

αγ

Streptococcus Habitat• Skin, mucous membranes, respiratory tract

and Gl/GU tracts, depending on species• 20% of children may carry GAS in their

pharynx during winter months.• S. pneumoniae is commonly isolated from the

respiratory tract of asymptomatic carriers.• Enterococci in gut flora are are important

pathogens in hospitals where they areselected by high antibiotic usage.

• Organisms spread by droplets, direct contactand fomites.

10

Lab Characteristics• Morphology and Gram stain

– Gram-pos. cocci 0.7 - 0.9 µm– pairs or chains

• Catalase-negative• Most grow on sheep blood agar• Aerobic or anaerobic• Enhanced by CO2

• Antigenic grouping• Biochemical identification

S. pyogenes in blood

S. pneumoniae in sputum

Antigenic Structure & VirulenceFactors of S. pyogenes

• Hyaluronic acid capsule- antiphagocytic

• Hyaluronidase - tissuepenetration

• Group specific cell wallantigen distinguishesfrom B,C,D,F,G, etc.

• Beta hemolytic

11

Antigenic Structure & VirulenceFactors of S. pyogenes

• M Protein– Virulence factor present on pilus with teichoic acid– Organisms lacking it are readily opsonized and

phagocytized– Binds fibrinogen, fibrin & degradation products

forming dense coating on the organism's surface,blocking complement

– Antibody against M protein is an importantprotective mechanism, but repeated infections withstrains possessing one of over 80 differentserotypes can occur

– Autoantibody target-Acute Rheumatic Fever

Antigenic Structure & VirulenceFactors of S. pyogenes

• Erythrogenic Toxin “Scarlet Fever”• Streptokinases

– transform plasminogen to plasmin– digest fibrin

• DNAase– depolymerizes DNA

antibody used to follow pyoderma• Hemolysins “Streptolysins”

– Important immunogens– Antibody against streptolysin O used

to follow course of pyoderma– Streptolysin S - β hemolysis

Strep. cellulitis

12

Antigenic Structure & VirulenceFactors of S. pyogenes

• Protein F - facilitatesattachment by bindingfibronectin

• Protein G - binds Fcportion of antibody

• Diphosphopyridinenucleotidase (DPNase)– enzyme kills WBCs

• C5a peptidase

Erysipelas

S. agalactiae “Group B”

• Pathogen of newborns& older debilitatedadults

• Antibody against type-specific capsularantigen is protective

• CAMP Test - interactionof CAMP factor(sphingomyelinase) withβ hemolysin of S.aureus to potentiatehemolytic activity

• Hippurate hydrolysis

GBS

S. aureus

13

Diseases Due to β Hemolytic StreptococciGroup Species Disease

A S. pyogenes Pharyngitis, impetigo,

cellulitis, erysipelas,

scarlet fever,

necrotizing faciitis,

rheumatic fever,

glomerulonephritis

B

S. agalactiae

Neonatal sepsis,

pneumonia, meningitis,

OB/GYN infections,

Bacteremia, UTI

C

S. equi, S. dysgalact iae,

& others

Bacteremia,

pneumonia,

endocarditis,

abscesses, pharyngitis,

wound infections

D

S. bovis

Endocarditis,

bacteremia in cancer

patients

F

S .anginosus

Cervicofacial

abscesses, bacteremia,

osteomyelit is

S. pneumoniae• Asymptomatic carriage• Colonization precedes disease• Most common cause of:

– community acquired pneumonia (bacteremia)– otitis media (children)– meningitis

• Species-specific C polysaccharide• No Lancefield antigen• 90 capsular serotypes• Alpha hemolytic• Bile soluble, optochin susceptible• Often require CO2 for growth Optochin test

14

S. pneumoniae Virulence Factors• Antiphagocytic capsule – immunogen• PspA: inhibits opsonization• Autolysin – release cell components• Pneumolysin

• Cytotoxic – inhibit cilia, wbcs• lyses RBCs• activates classic complement path.• stimulates cytokines → tissue

damage & purulent inflammation• Hydrogen peroxide - tissue damage• Surface protein adhesins• Neuraminidase• IgA protease• Peptidoglycan

– activate alternate complement– cytokine release

• Transformation– antibiotic resistance• Intracellular invasion Capsule Quellung Reaction

sputum - pneumonia

Fatal Pneumococcal Meningitis

15

Pneumococcal Vaccine

• 23 valent polysaccharide vaccine - adults• 7 valent conjugate vaccine - children

Viridans streptococci• Most human strains are commensals of the

oral cavity & upper respiratory tract• Alpha hemolysis• Do not have Lancefield group antigens• Differentiate species biochemically• Usually of low pathogenicity• Important causes of endocarditis• Dental caries (S. mutans) → dextran from

glucose

16

Enterococcus• At least 12 species• Usually non-hemolytic• E. faecalis most common• Distinguish from streptococci by:

– esculin hydrolysis– growth in 6.5% NaCl– PYR hydrolysis (Group A β strep. are +)

• Enteric flora• Opportunist - nosocomial pathogen• Intrinsic antimicrobial resistance• E. faecium - vancomycin-resistance• Abscesses, urinary tract, endocarditis,

abdominal/pelvic, bacteremia, wound infections

Listeria monocytogenes• Gram-positive coccobacilli• Catalase positive• Weakly β hemolytic• Facultatively intracellular• Motile• Attachment factors,

hemolysins, phospholipases• Diseases

– foodborne (dairyproducts)

– meningitis– neonatal infections

17

Key PointsListeria monocytogenes

• Gram-positivebacilli/coccobacilli

• Catalase-positive• Motile• Esculin positive• β hemolytic

Positive motility test

Corynebacterium• Gram-positive, curved pleomorphic rods• “Chinese Letters”• Aerobic/facultatively anaerobic• Grow readily on SBA• Need selective media (cystine-tellurite)

to detect and ID• Catalase & oxidase positive• Usually non-motile• Commensals on skin• Pathogens

– C. jeikeium– C. diphtheriae

18

Diphtheria• Toxin acts locally on mucous

membranes of respiratory tract• Adherent pseudomembrane of

fibrin, bacteria, epithelial andphagocytic cells impairs breathing

• Toxin spreads hematogenously toheart, CNS, and adrenals

• Cytotoxic effect mediated byinterfering with proteinbiosynthesis by inactivating EF-2

• Spreads by droplet• Protect by vaccination - DaPT

(diphtheria toxoid)• < 5 cases/yr in US

Bacillus• Endospore-forming• Aerobic/facultative anaerobic• Gram-positive bacilli• Can be gram-variable• Often β hemolytic & motile

– (except B. anthracis)• Usually catalase positive• Skin flora - lab contaminants• Food poisoning - B. cereus• Ocular infections – B. cereus• Anthrax – B. anthracis

Gray, spreading β-hemolytic colonies on SBA

19

Bacillus Endospores

B. anthracis: Anthrax• Rare in US due to control in animals• Enzootic in middle east• Transmitted by contact with animal products• Spores remain infectious for years• Usually cutaneous inoculation - slow healing ulcer,

bacilli spread to lymphatics and bloodstream– 20% mortality if untreated– Respiratory anthrax is usually fatal even if treated– GI anthrax also occurs in some Asian countries

• Vaccine given to military• Excellent biological warfare agent

20

Anthrax Pathogenesis

• Poly-D-glutamic acid (protein) capsule – antiphagocytic• 3 component exotoxin

Protective antigen (PA) - binds to cells, forms channelthat permits EF and LF to enterEdema factor (EF) – adenyl cyclase causes fluid toaccumulate at the site of infection and inhibit immunefunctionLethal factor (LF)-disrupts cell's functions, stimulates TNF-alpha and IL-1-beta, kills infected cells

Anthrax Vaccine

• Made from avirulent,nonencapsulated B. anthracis strain

• Requires series of injections andannual boosters

• Used in military