Chapter 22 Streptococcus, Enterococcus and Pneumococcus

STERPTOCOCCUS,
ENTEROCOCCUS &
PNEUMOCOCCUS
Learning objectives
At the end of the session, the students will be able to
• Describe morphology and antigens
• Classify
• Describe Pathogenesis & Clinical features
• Choose appropriate lab diagnosis and interpret the
results
• Describe prevention and treatment
Essentials of Medical Microbiology by Apurba S Sastry
© 2018, Jaypee Brothers Medical Publishers
STREPTOCOCCUS

STREPTOCOCCUS
• General Features:
- Gram positive cocci in pairs and chains
- Catalase negative

History
- Billroth coined the term ‘streptococci’ (streptos
meaning twisted or coiled),
- Ogston differentiated them from staphylococci
- Rosenbach coined the species S. pyogenes

Classification of Streptococcus

STREPTOCOCCUS PYOGENES

VIRULENCE FACTORS.. Cell wall antigens
• Lipoteichoic acid – adhesion
• M protein
- Opsonization
- Strep. Toxic Shock Syndrome
- Ig to class 1 M protein
Rheumatic fever

VIRULENCE FACTORS.. Toxins
Streptolysin-O (SL-O) Streptolysin-S (SL-S)
Oxygen labile , Heat labile Oxygen stable, Serum soluble
Hemolysis only in deep colonies Hemolysis on surface of blood
(pour plate) agar
Cytotoxic for neutrophils, Leucocidal
platelets and cardiac tissue.
Strongly antigenic Not antigenic
Antistreptolysin-O antibodies Not useful for serological
(ASO) -for retrospective diagnosis diagnosis of streptococcal
of streptococcal infections infections.

VIRULENCE FATCORS.. Toxins
Haemolysins
• Streptolysin-O is structurally and functionally similar to-
 Tetanolysin of Clostridium tetani
 Pneumolysin of S.pneumoniae
 Theta toxin of Clostridium perfringens
 Listeriolysin O of Listeria
 Cereolysin of Bacillus cereus
Streptococcal pyrogenic exotoxin (SPE)
• Causes - scarlet fever, necrotizing fasciitis & streptococcal toxic
shock syndrome.
• Antigenic subtypes- SPE-A,B and C
• SPE-A and C are bacteriophage coded, superantigens
• SPE-B is chromosomally mediated.
• Dick test-SPE was previously called as erythrogenic or scarlet
fever toxin because its intradermal injection in susceptible
children produced local erythema indicating susceptible to
scarlet fever.
VIRULENCE FATCORS.. Enzymes
• Streptokinase (fibrinolysin)-
• Activates plasminogen to plasmin  breaks down
the fibrin barrier  spread of infection.
• Antibodies to streptokinase - retrospective diagnosis
• Therapeutic use- myocardial infarction and other
thromboembolic disorders.

• Streptodornase (DNase)-
• Liquefying the thick pus - responsible for the serous nature of
streptococcal exudates
• Therapeutic use- to liquefy the thick exudates in empyema
cases.
• Subtypes -B is most antigenic
• Diagnostic use- Anti-DNase B antibodies - retrospective
diagnosis of skin infections (pyoderma) and acute
glomerulonephritis where ASO titre is low
• Hyaluronidase (spreading factor)- breaks down the
hyaluronic acid of the tissues- spread of infection
• NADase- antigenic and leucotoxic.
• Serum opacity factor- Lipoproteinase - opacity when
applied on agar gel containing serum.
• SpyCEP- Serine protease - inactivates interleukin 8
(neutrophil chemoattractant)
• C5a peptidase- Serine protease -Cleaves C5a (neutrophil
chemoattractant)
Streptococcal Suppurative Infections
• Pharyngitis • Scarlet fever
- GAS is MC cause in children - SPE A, B & C
- Erythema and swelling of - Rash with sandpaper
pharyngeal mucosa with
purulent exudate
feel
- Complications due to - Rash in skin folds
contiguous spread, through - Strawberry tongue
blood and lymph
• Impetigo
- superficial infection of the
skin – face & legs
- Children, tropical climates,
poor hygiene, colonization
of group A Streptococcus Red papules vesicles 
and trauma pustular lesions  thin papery
- Painless & No fever honeycomb-like crusts
• Erysipelas:
- Form of cellulitis
- Tender, bright red, swollen and
indurated peau d'orange texture
of involved skin -Superficial blebs
or bullae
- Fever and chills
- Malar area of the face & legs
- Recurrences involving the same
site.
• Necrotizing Fasciitis
- Aka- hemolytic streptococcal
gangrene
- GAS - MC cause (60%) – Flesh
eating bacteria - Pyrogenic
exotoxins • Dusky Skin or mottled
• Source of the infection erythema and anesthetized
- Trauma & GIT breach extensive necrosis of
• Invasion of Fascia &Muscles subcutaneous tissue, fascia
Essentials of Medical Microbiology by Apurba S Sastry and muscle
Streptococcal Non-Suppurative Lesions
• Acute rheumatic fever Streptococcal Ag Mammalian Ag
• Post-streptococcal Cell wall M protein Myocardium
glomerulonephritis (PSGN) Cell wall C Cardiac valves
• Guttate psoriasis carbohydrate
• Reactive arthritis Cytoplasmic Glomerular vascular
• Pediatric Autoimmune membrane intima
Neuropsychiatric Disorders
Peptidoglycan Skin antigens
• Associated with Streptococcus
Hyaluronic acid Synovial fluid
pyogenes (PANDAS)
• Acute Rheumatic Fever
• Previous streptococcal pharyngitis
• Pathogenesis
- Autoimmune theory: Antibody cross reaction &
- Cytotoxicity theory
• Clinical manifestations
- Affects heart, joints, and skin. Modified Jones criteria
• Repeated attacks common – Long-term penicillin prophylaxis
• ASO titer
Revised Jones Criteria..
Criteria Manifestations
Major manifestations Subcutaneous nodules
(SPACE) Pancarditis
Arthritis (migrating polyarthritis)
Chorea (CNS manifestation)
Erythema marginatum (skin lesion)
Minor manifestations Clinical: fever, arthralgia
Laboratory: elevated ESR and C-reactive
protein
ECG: Prolonged P-R interval
tials of Medical Microbiology by Apurba S Sastry
Revised Jones Criteria..
Criteria Manifestations
Supporting evidence Elevated ASO, or
(of previous A positive throat culture, or
streptococcal Rapid antigen test for GAS, or
infection) Recent scarlet fever
Rheumatic fever is Two major manifestations or

diagnosed if: one major and two minor manifestations
plus evidence of previous streptococcal
infection
• Post-streptococcal Glomerulonephritis (PSGN)
- 2–3 weeks following pyoderma - (M types–49, 53-55
and 59-61) or rarely pharyngitis (M-types 1 and 12)
• Antigen antibody complex deposition on the
glomerular basement membrane complement
activation urine retention and renal insufficiency
edema, hypertension, hematuria and proteinuria
Rheumatic Fever v/s Acute Glomerulonephritis
Feature ARF PSGN
Prior history of Pharyngitis strains Mainly pyoderma, or
infection with rarely pharyngitis
strains
Serotype Most strains of GAS Pyodermal strains-
49,53-55, 59-61&
Pharyngitis strains- 1,12
Immune response Marked Moderate
Complement level Unaltered Low (due to
depositionin glomeruli)

Rheumatic Fever v/s Acute Glomerulonephritis
Feature ARF PSGN
Genetic Present Absent
susceptibility
Repeated attack Common Uncommon
Penicillin prophylaxis Indicated Not indicated
Course Progressive Spontaneous
resolution
Prognosis Variable Good
Hypersensitivity Type II Type III
reaction

Lab Diagnosis...
• Specimens:
- Throat swab, pus swab,
exudates and blood
- Pike’s transport media
• Microscopy
- Pram positive cocci in
chains
Lab Diagnosis... Culture
• Aerobe & Fastidious
• Blood Agar – Beta
hemolytic pinpoint colonies
• Liquid medium – Granular
turbidity
• Selective Medium
- Crystal violet blood agar
- PNF medium
Biochemical reactions
• Catalase – positive
• Bacitracin - sensitive

Serologic Classification
• Lancefield Grouping • Griffith Typing
- Based on C-antigen - Based on M protein
- Lancefield’s acid extraction - 100 serotypes
- Fuller’s method
- Rantz and Randall method
- Emm Typing – based on
- Maxted’s method
gene coding for M
protein
SEROLOGY
• ASO (Anti-streptolysin O) • Anti-DNase-B antibodies –
antibodies - Titre >300-350 units/ml is
- Titer >200 Todd unit/ml in diagnostic of PSGN and
most of the streptococcal pyoderma
infections except • Anti-hyaluronidase
pyoderma and PSGN. antibodies
- detected by latex • Anti-streptokinase
agglutination test. antibodies
TREATMENT
Conditions Treatment recommended
Pharyngitis Benzathine penicillin G, IM single dose;
or Oral penicillin V for 10 days
Erysipelas/Cellulitis Mild- Procaine penicillin
Severe- Penicillin G
Necrotizing fasciitis Surgical debridement (most crucial) + Penicillin G + Clindamycin
Pneumonia & empyema Penicillin G + drainage of empyema
Streptococcal TSS Penicillin G + Clindamycin + immunoglobulin (to SPE)
Benzathine penicillin G, IM single dose;
or Oral Penicillin V for 10 days
Long term maintenance therapy- with penicillin G monthly
Rheumatic fever
 5 yr or until 21 yr of age, (without carditis),
 10 yrs (with carditis),
 up to 40yr of age / lifelong (with residual heart disease)
OTHER BETA HEMOLYTIC STREPTOCOCCI

S.agalactiae
• Group B Streptococcus
• Carriage in vagina and rectum in 30% women
• Neonatal sepsis & meningitis
• Pregnancy  peripartum fever
• Cellulitis and soft tissue infections urinary tract
infection, pneumonia, and endocarditis – in diabetics,
cancer patients
Early v/s Late GBS Disease
Characteristics Early-onset disease Late-onset disease
Age of onset 0–6 days of birth 7–90 days of birth
Increased risk following Prematurity and prolonged Not associated
obstetric complications labor
Mode of transmission to During or before birth from Contact with a colonized
the baby the colonized maternal mother and nursing
genital tract personnel
Common clinical Pneumonia and/or Bacteremia&meningitis
manifestations respiratory distress (most common)
syndrome followed by
meningitis
Common serotypes Ia, III, V, II, Ib III predominates
Case fatality rate 4.7% 2.8%

S.agalactiae
• Lab Diagnosis
• CAMP positive: CAMP
factor (named after the
discoverers: Christie,
Atkins-Munch-Petersen)

S.agalactiae
• Other Tests: • Treatment
- Hippurate hydrolysis test - Penicillin or higher penicillins
positive
- Bacitracin resistant • Prevention
- PYRtest negative - Screening at 35-37 wks of
- Orange pigment - Islam’s pregnancy
medium - Ampicillin or penicillin to
- Mucoid β haemolytic colonies carrier mothers

GROUP C STREPTOCOCCI
• Common animal pathogen. Human infections rare
• S.equi, S.equisimilis, S.dysgalactiae, S.zooepidermicus
• S.equisimilis - pharyngitis especially epidemic food-borne
pharyngitis after ingestion of contaminated products (milk)
• Others- skin and soft tissue infections, osteomyelitis,
pneumonitis, infective endocarditis, bacteremia and septicemia,
meningitis, epiglottitis, pericarditis, urinary tract infections, and
puerperal sepsis.
• S.equisimilis - source of streptokinase
GROUP F STREPTOCOCCI
- Minute streptococci,
- Grow poorly on blood agar
- occasionally cause suppurative infection.
• Antibodies to Streptococcus MG for diagnosis of
primary atypical pneumonia (caused by Mycoplasma
pneumoniae)

Other β Hemolytic Streptococci
• GROUP G STREPTOCOCCI
• Throat commensals
• Occasionally causing puerperal sepsis, neonatal infection, skin
and soft tissue infections, tonsillitis, and endocarditis.
•
• GROUP D STREPTOCOCCI
• common group D lipoteichoic acid antigen
• Group D streptococci comprises of enterococci & non-
enterococci (S.bovis and S.equinus)
ENTEROCOCCUS

Enterococcus – general Features
• Initially grouped under group D Streptococcus,
reclassified as a separate genus Enterococcus
• Normal flora of human intestine, biliary tract and to
lesser extent vagina and male urethra
• Increasingly becoming important agents of human
disease especially in hospitals mainly because of their
resistance to antibiotics
• Common species - E.faecalis & E.faecium
Virulence Factors
• Cytolysin/hemolysin
• Aggregation substances or pheromones - clumping of
adjacent cells to facilitate plasmid exchange (transfers drug
resistance)
• Extracellular surface protein (ESP) – adhesion to bladder
mucosa
• Common group D lipoteichoic acid antigen - cytokine
release such as tumor necrosis factor α (TNFα)
• Coccolysin - inactivates endothelin, a vasoactive peptide.
Clinical Manifestations
• Urinary tract infections (cystitis, urethritis, pyelonephritis
& prostatitis)
• Bacteremia and mitral valve endocarditis (in intravenous
drug abusers)
• Intra-abdominal, pelvic and soft tissue infections
• Late-onset neonatal sepsis and meningitis
• Infection on burn surface.

Laboratory Diagnosis
• Gram-positive oval cocci
arranged in pairs, arranged at
an angle to each other
(spectacle-shaped
appearance)
• Blood agar - non-hemolytic
translucent colonies
• MacConkey Agar – Tiny
magenta pink
• Nutrient agar: It grows poorly
• Bile aesculin hydrolysis test is positive 
• PYR (Pyrrolidonyl-beta-naphthylamide)
test is positive
• Can grow in presence of extremes of
conditions such as—6.5% NaCl, 40% bile, pH
9.6, 45°C and 10°C
• Heat tolerance test - can survive 60°C for 30
minutes
Enterococci v/s Group D streptococci
Characteristics Enterococci Group D streptococci
Group specific D antigen Present Present
Bile aesculin hydrolysis test Positive Positive
In presence of - 6.5% NaCl, pH Grows Does not grow
9.6, at 450C, at 100C
PYR test Positive Positive

Drug resistance Marked Uncommon
Normal GIT flora Predominant Less
Pathogenicity Marked Less
Antibiotic Resistance
• Resistant to Penicillins, cephalosporins, Sulfonamides,
Aminoglycosides and cotrimoxazole
• Resistance to Penicillins and Aminoglycosides can be
overcome by combination therapy – Synergistic
• VRE – Vancomycin Resistant Enterococcus
- Indian prevalence – 5-10%
- Van gene – alters target at cell wall. Van A & Van B more
prevalent globally
VIRIDANS STREPTOCOCCI

• Commensals of mouth • Members
and upper respiratory - S.mutans
tract - S. Intermedius
• Usually non-pathogenic - S. anginosus
- S.salivarius
- S.sanguis
- S.milleri
• Dental caries
- S. mutans - Dietary sucrose to acid and dextrans  Acid damages
dentine, dextran binds together food debris, mucus, epithelial
cells &bacteria to produce dental plaques
• Subacute bacterial endocarditis (SABE)
- Oral Viridans streptococci MC cause  Transient bacteremia while
chewing, tooth brushing & dental procedures endocarditis
• S. milleri group (includes S. intermedius, S. anginosus, and S.
Constellatus) -suppurative infections (abscesses of brain &
abdominal viscera)
LAB DIAGNOSIS
• Gram stain – Gram
positive cocci in chains
• Blood Agar – Alpha

hemolytic colonies

Differences between Viridans Streptococci & Pneumococcus
Feature S.pneumoniae Viridans streptococci

Morphology Lanceolate or flame shaped Round / oval
Arrangement Gram positive cocci in pairs Gram positive cocci in long
chains
Capsule Present Absent
Colony on blood agar Draughtsman or carom coin Convex shaped colony
Liquid medium Uniform turbidity Granular turbidity
Bile solubility Soluble in bile Insoluble in bile
Inulin fermentation Fermenter Non fermenter
Optochin Sensitive Resistant
Mice Pathogenicity Pathogenic Non-pathogenic
STREPTOCOCCUS PNEUMONIAE
(PNEUMOCOCCUS)
Virulence Factors and Pathogenesis
• Capsular polysaccharide
- Protects the cocci from phagocytosis
- soluble - diffuses into culture media, tissue and exudates
(soluble specific substance)
• C carbohydrate antigen(C-polysaccharide or C-substance) -
Species specific
• Pneumolysin - inhibits neutrophil chemotaxis and phagocytosis
• Autolysin - Autolysis of cells - enhanced by bile salts & other
surface active agents bile solubility and draughtsman colony
Other virulence factors
• Pneumococcal surface protein A (PspA) – prevents
complement activation
• IgA protease - cleaves IgA in respiratory mucosa, thus
facilitates entry
• Pneumococcal surface protein C (PspC) /choline-binding
protein A (CbpA) – Binds to factor H and accelerates the
breakdown of C3 complements
• Adhesins - sialidase (neuraminidase) & pneumococcal
surface adhesin A (PsaA).
• Colonize human nasopharynx spread either via
bloodstream to distant sites (e.g. brain, joint, bones &
peritoneal cavity) or spread locally to cause otitis media
or pneumonia.
• Lobar pneumonia
- MC cause of lobar (alveolar) pneumonia
- Known to progress to bacteremia and invasive disease
- Empyema & parapneumonic effusion
• Invasive pneumococcal disease
• Defined as an infection confrimed by isolation of pneumococci from
normally sterile site
• Blood stream infection
• Pyogenic meningitis – leading cause of meningitis in all ages (except in
neonates)
• Others - cause osteomyelitis, septic arthritis, endocarditis, pericarditis,
primary peritonitis, rarely, brain abscess & hemolytic-uremic syndrome.
• Noninvasive manifestations - otitis media & sinusitis (MC cause)

Epidemiology
• Source - upper respiratory tract of humancarriers (less
often patients)
• Carrier rate - >90% of children of 6 m to 5 yrs – in
nasopharynx
• Mode of transmission – inhalation droplet nuclei
• Infection usually leads to colonization and carrier state.
• Disease results only when the host resistance is lowered
due to presence of associated risk factors.
Risk Factors
• Children (<2 years)
• Splenectomy, sickle cell disease & other
hemoglobinopathies
• Underlying comorbid diseases - chronic lung, heart, kidney
and liver disease, cochlear implants, diabetes mellitus
&immunosuppression
• Children - Serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F
• Adults - Serotypes 1–8
• Virulent serotypes - Serotype 3 followed by 7
• Specimen Collection
- Sputum, cerebrospinal fluid
(CSF), pleural fluid and other
sterile body fluids, Blood
culture
• Microscopy
- Lanceolate-shaped Gram
positive cocci in pairs
- India ink - capsule
CULTURE
• Enriched media,5–10% CO2
• Blood agar – Alpha
hemolytic draughtsman-
shaped or carom coin-
shaped colony
• Chocolate agar – bleeching
effect
• BHI broth – Uniform
turbidity  clearing
Biochemical Identification
• Catalase negative
• Bile sensitivity
- Plate and Tube method
• Optochin sensitive
• Inulin fermented
• Quellung reaction

Antimicrobial Susceptibility Test
• Oxacillin - disk diffusion • Treatment
test • Penicillin – DOC
- Sensitive  sensitive to • Alternatives
Penicillin, ceftriaxone
- Cephalosporins
and other β lactams
- Vancomycin
- Resistant  MIC testing
for individual β lactams - Quinolones
should be done
Drug Resistance in Pneumococcus
• Penicillin Resistance • MDR Pneumococcus
Alteration of PBP to Resistant to
PBP2a - Penicillins
- Erythromycin
- Tetracycline
- Clindamycine
- Sulfonamides
Prevention
• Vaccines
- PPSV23 - 23-valent pneumococcal polysaccharide
vaccine
- PCV13 - pneumococcal conjugate vaccine

Pneumococcal Vaccines
PPSV23 PCV13
Brand Pneumovax 23 Prevnar13
Coverage 70-80% invasive serotypes Most serotypes in children, 30-
in adults 40% in adults
Immunogenicity Less More
Protection Short term (3-5 yrs) Longer
Herd immunity Yes No
Carriers No mucosal immunity. No Provides mucosal immunity.
effect on carriers Eradicate carriage
Cost Less More
Indication >65yrs, Adults with risk Children, >65yrs, Adults with
factors risk factors
QUICK ASSESSMENT

MCQs
• Serotyping of • Streptococcus pyogenes
Streptococcus pyogenes is can be differentiated from
based on which of the Streptococcus agalactiae
following protein? by:
a. M protein a. Optochin sensitivity
b. T protein b. Bacitracin sensitivity
c. R protein c. Polymyxin sensitivity
d. Carbohydrate antigen d. Novobiocin sensitivity
MCQs
• C AMP test is useful in • Which is a
identification of: poststreptococcal
a. S. Pyogenes sequelae:
b. S. Agalactiae a. Acute rheumatic fever
c. Viridans streptococci b. Cellulitis
d. S. pneumoniae c. Pharyngitis
d. Impetigo
MCQs
• Neonatal meningitis • Carrom coin
acquired through appearance of colonies
infected birth canal is is seen for:
due to:
a. S. pyogenes
a. S. pyogenes
b. Viridans streptococci
b. Viridans streptococci
c. S. agalactiae
c. S. agalactiae
d. S. pneumoniae
d. S. pneumoniae
MCQs
• Which is not a property
of S. pneumoniae?
a. Bile solubility
b. Animal pathogenicity in
mice
c. Growth in presence of
40% bile
d. Optochin sensitivity
THANK YOU...!


Chapter 22 Streptococcus, Enterococcus and Pneumococcus

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STERPTOCOCCUS,

Essentials of Medical Microbiology by Apurba S Sastry

Essentials of Medical Microbiology by Apurba S Sastry

Essentials of Medical Microbiology by Apurba S Sastry

Essentials of Medical Microbiology by Apurba S Sastry

Essentials of Medical Microbiology by Apurba S Sastry

Essentials of Medical Microbiology by Apurba S Sastry

Essentials of Medical Microbiology by Apurba S Sastry

Essentials of Medical Microbiology by Apurba S Sastry

Rheumatic fever is Two major manifestations or

Essentials of Medical Microbiology by Apurba S Sastry

Essentials of Medical Microbiology by Apurba S Sastry

Essentials of Medical Microbiology by Apurba S Sastry

Essentials of Medical Microbiology by Apurba S Sastry

Essentials of Medical Microbiology by Apurba S Sastry

Essentials of Medical Microbiology by Apurba S Sastry

Essentials of Medical Microbiology by Apurba S Sastry

Essentials of Medical Microbiology by Apurba S Sastry

Essentials of Medical Microbiology by Apurba S Sastry

Essentials of Medical Microbiology by Apurba S Sastry

PYR test Positive Positive

Essentials of Medical Microbiology by Apurba S Sastry

• Blood Agar – Alpha

Essentials of Medical Microbiology by Apurba S Sastry

Feature S.pneumoniae Viridans streptococci

• Noninvasive manifestations - otitis media & sinusitis (MC cause)

Essentials of Medical Microbiology by Apurba S Sastry

Essentials of Medical Microbiology by Apurba S Sastry

Essentials of Medical Microbiology by Apurba S Sastry

Essentials of Medical Microbiology by Apurba S Sastry

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• Noninvasive manifestations - otitis media & sinusitis (MC cause)