STREPTOCOCCUS,

ENTEROCOCCUS, AND
OTHER CATALASE-
NEGATIVE, GRAM-
POSITIVE COCCI
CLINICAL BACTERIOLOGY
Learning Outcomes

■ Compare the general characteristics and isolation of streptococci and similar

organisms.

■ Explain the Lancefield classification of streptococci.

■ Apply the knowledge of hemolytic patterns on sheep blood agar in the

identification of streptococcal isolates..

■ Describe the virulence factors and clinical infections associated with Streptococci

■ Discuss how to identify Streptococci from a clinical sample

 Family
Streptococcacea  Genus Streptococcus

e  pathogenic and saprophytic

streptococci
Classification  Genus Enterococcus
Domain: Prokaryotes  fecal streptococci that were
Kingdom: Bacteria associated with the human
and animal gastrointestinal
Phylum: Firmicutes tract (GIT), and with some
Class: Bacilli fermented foods
Order: Lactobacillales  formerly group D streptococci
Family: Streptococcaceae  Now under Family
Genus: Streptococcus Enterococcaceae
 Genus Lactococcus
 formerly group N streptococci
  catalase-negative
General  gram-positive cocci that are
Characteristic usually arranged in pairs or
chains (broth)
s of  Cells are more elongated than
Streptococci spherical
 Generally considered
and similar facultative anaerobes; some
capnophilic; some obligate
organisms anaerobic
 Ferments carbohydrates
without gas production
 Grows poorly on nutrient agar
 Grows best on enriched media
 Cell Wall Structure
General  possess a typical gram-
Characteristic positive cell wall consisting
s of of peptidoglycan and
teichoic acid.
Streptococci  have C CARBOHYDRATE
and similar (polysaccharide); (except
for many of the viridans
organisms group)
 can be used to classify
an isolate serologically.
 developed in the 1930s
by Rebecca Lancefield
 General
Characteristic
s of Hemolysis Pattern
Streptococci  can produce numerous
and similar exotoxins that damage
intact red blood cells (RBCs)
organisms  Basis for classification
Type of
Hemolysis
Lancefield Grouping

■ a serological method for classifying streptococci into one of 20 groups

(designated by a letter: A, B, C, D…T) based on the presence of
polysaccharide and teichoic acid antigens in the bacterial cell wall

– Organisms in group A possess the same antigenic C carbohydrate

– Organisms in group B have the same C carbohydrate, and so on

Clinically Significant Streptococci and
Streptococcus-like Organisms
Clinically Significant Streptococci
and
Streptococcus-like Organisms
STREPTOCOC
CUS
PYOGENES
Streptococcus pyogenes

■ Group A Streptococcus (GAS)

■ major human-specific bacterial
pathogen
■ causes a wide array of
manifestations
– mild localized infections
– life-threatening invasive
infections
Streptococcus pyogenes

■ General Characteristics
■ Microscopic: Gram-positive cocci in chains or pairs
■ Colony Morphology:
– forms pinpoint colonies on blood agar plates
– Large β-hemolysis
■ catalase-negative
■ oxidase negative
■ facultative anaerobe, grows best in 5 to 10% carbon dioxide
Streptococcus pyogenes
■ Pathogenesis and virulence factors
– Enters through respiratory tract or skin contact
– Either local disease or spread
– M protein - resist phagocytosis; adherence to mucosal
cells
– Lipoteichoic Acid - for attachment
– Fibronectin-binding Protein (Protein F) – for
attachment
– Hyaluronic acid capsule- inhibits phagocytosis;
weakly immunogenic
– Extracellular products
Streptococcus pyogenes
■ Pathogenesis and virulence factors
– Extracellular products:
■ Streptococcal Pyrogenic Exotoxins (erythrogenic)
– causes the rash of scarlet fever
– superantigen
■ Streptokinase
– Converts plasminogen to protease (plasmin) which lyses the fibrin clot
■ Streptolysin S
– Oxygen stable, lyses leukocytes, and is non-immunogenic
– responsible for hemolysis on SBA plates (aerobic)
■ Streptolysin O (SLO)
– Oxygen labile, lyses leukocytes and platelets, and highly immunogenic
– responsible for hemolysis on SBA plates (anaerobic)
■ Deoxyribonuclease (DNase) - antigenic
■ Hyaluronidase
– Spreads infection
Streptococcus pyogenes
■ Transmission – TSS
– spread from person to – Some less common
person infections
– Carriers do exist ■ Pneumonia
■ Meningitis
■ Clinical disease
■ Fasciitis
– “Strep throat” pharyngitis
– Complications
– Skin infections
■ Rheumatic heart disease
– Otitis
■ Glomerulonephritis
– Sinusitis
– Scarlet fever
Streptococcus pyogenes
■ “Strep throat” pharyngitis
– Most common
– S/S:
■ sudden-onset of sore throat;
odynophagia, fever (some –
scarlet fever)
■ Pharyngeal and tonsillar
erythema
■ Tonsillar hypertrophy with or
without exudates
■ Palatal petechiae
■ Anterior cervical
lymphadenopathy
– Common in 5-15 y.o.
– spread by droplets and close
contact
Streptococcus pyogenes
PYODERMAL INFECTIONS (skin
infections)
■ Impetigo (non-bullous)
■ Erysipelas
■ Cellulitis
■ Necrotizing fasciitis
Streptococcus pyogenes
PYODERMAL INFECTIONS (skin infections)
■ Impetigo
– red, itchy sores that break open and leak a clear fluid or
pus for a few days. Next, a crusty yellow or “honey-
colored” scab forms over the sore, which then heals
without leaving a scar.
■ Cellulitis
– infection that occurs in the subcutaneous tissues
– appears as a red, swollen, and painful area of skin that
is warm and tender to the touch
■ Erysipelas
– rare infection of the skin and subcutaneous tissues
– acute spreading skin lesion that is intensely
erythematous with a plainly demarcated but irregular
edge
Streptococcus pyogenes
NECROTIZING FASCIITIS
– rapidly progressing inflammation and necrosis
of the skin, subcutaneous fat, and fascia
– uncommon, BUT is life threatening
STREPTOCOCCAL TOXIC SHOCK SYNDROME
– condition in which the entire organ system
collapses(liver, kidney, lung, blood) leading to
death
– initial streptococcal infection is often severe
– begins with influenza-like symptoms, including:
Fever, Chills, Myalgia, Nausea, Vomiting
– Superantigens lead to overstimulation of the
immune response
Streptococcus pyogenes
SCARLET FEVER
■ occurs with group A strep pharyngitis or follow
a group A strep pyodermal or wound
infections.
■ also called scarlatina (scarlatiniform rash):
– Erythematous rash that blanches on
pressure
– Sandpaper quality
– Accentuation of the red rash in flexor
creases, termed “Pastia’s lines”
– Begins on the trunk, then quickly spreads
outward, usually sparing the palms, soles,
and face (some with circumoral pallor)
– A yellowish white coating with red papillae
may initially cover the tongue. The
eventual disappearance of the coating can
result in a “strawberry tongue.”
– persists for about one week followed by
desquamation
Streptococcus pyogenes
POSTSTREPTOCOCCAL SEQUELAE OR COMPLICATIONS:

■ Rheumatic Fever

■ Acute Glomerulonephritis
Streptococcus pyogenes
POSTSTREPTOCOCCAL SEQUELAE OR COMPLICATIONS:
■ Rheumatic Fever
– fever and inflammation of the heart, joints, blood vessels, and
subcutaneous tissues.
– Attacks usually begin within 1 month after infection
– Could lead to chronic, progressive damage to the heart valves
– Repeated infections can produce further valve damage
– Could lead to Rheumatic Heart Disease (permanent damage)
Streptococcus pyogenes
POSTSTREPTOCOCCAL SEQUELAE OR COMPLICATIONS:
■ Acute Glomerulonephritis
– occurs after a cutaneous or pharyngeal infection
– more common in children than in adults
– immunologically mediated:
■ Circulating immune complexes are found in the serum of patients
■ these complexes deposited in the glomeruli + complement fixed
– inflammatory response causes damage to the glomeruli, resulting
in impairment of kidney function.
Streptococcus pyogenes

■ Laboratory diagnosis

– Specimen: properly collected THROAT/SKIN SWAB

– Specimen Processing:
■ Inoculate and streak SBA or Streptococcus-Selective Agar (SSA)

■ Add Bacitracin disc

■ incubate 35°C either in ambient air or under anaerobic

conditions

■ Incubate for up to 48 hours before reporting as negative

Streptococcus pyogenes
■ Laboratory diagnosis
– Growth Work-up:
■ Gram Stain: GPC in chains
■ Colony: pinpoint/small, transparent, smooth,
beta-hemolytic
■ Bacitracin (diagnostic disc “A”) susceptible
■ L-pyrolidonyl arylamidase (PYR) positive
■ Latex test or other grouping test
Streptococcus pyogenes
Streptococcus pyogenes
Laboratory diagnosis
■ Growth Work-up:
– For blood and sputum culture
■ Serologic tests not performed
■ Additional tests performed:
– Hippurate hydrolysis
– CAMP test
– bile esculin test
– growth in 6.5% sodium
chloride (NaCl) broth
Streptococcus pyogenes

■ Susceptibility testing and treatment

– All isolates are penicillin susceptible

– Susceptibility testing not appropriate in almost

all settings
– Exceptions are penicillin-allergic patients
Streptococcus pyogenes
Streptococcus pyogenes

Laboratory diagnosis
■ Immunologic tests (serum sample)
– ASO titers
– anti-Dnase titers
– Antistreptokinase titers
– antihyaluronidase titers
Streptococcus pyogenes Review

■ Microscopic morphology
■ Colony Morphology
■ Culture requirements
■ Identification
■ Clinical infections
STREPTOCOCC
US
AGALACTIAE
Streptococcus agalactiae

■ Group B streptococcus (GBS)

– Have Group B–specific antigen, an acid-stable polysaccharide
– the only species in group B but have 9 serotypes
■ Normal flora in genital tract and gastrointestinal tract
■ Clinical significance
– Neonatal sepsis and meningitis
– UTI
– Pneumonia in elderly
Streptococcus agalactiae

■ General Characteristics
■ Microscopic: Gram-positive cocci in chains or pairs
■ Colony Morphology:
– forms medium-sized, grayish white mucoid colonies on SBA
– β-hemolytic (small)
■ catalase-negative
■ oxidase negative
■ facultative anaerobe, grows best in 5 to 10% carbon dioxide
Streptococcus agalactiae
Pathogenesis and virulence factors
■ Capsule
– Prevent phagocytosis but is ineffective after opsonization
■ Sialic Acid
– most significant component of the capsule
– critical virulence determinant
– weakly immunogenic and can inhibit activation of the
alternative complement pathway
■ Other products:
– hemolysin, CAMP factor, neuraminidase, DNase,
hyaluronidase, and protease.
Streptococcus agalactiae

CLINICAL INFECTIONS

■ Neonatal Disease

– Early-onset neonatal disease (<7 days old)

– Late-onset (7 days to 3 months)

■ Non-pregnant Adult

■ Elderly infections
Streptococcus agalactiae

CLINICAL INFECTIONS
■ Early-onset neonatal disease (<7 days old)
– vertical transmission from mother
– accounts for about 80% of the clinical cases in newborns
– Associated with obstetric complications, prolonged rupture of
membranes, and premature birth
– often manifests as pneumonia and sepsis
– High mortality rate
* Pregnant women are screened for presence of GBS in their vagina and
rectum
Streptococcus agalactiae

CLINICAL INFECTIONS
■ Late-onset (7 days to 3 months)
– meningitis is acquired horizontally, some as
nosocomial infection
– manifests as meningitis and sepsis
– Mortality rate is less compared to EOD but still high
Streptococcus agalactiae

CLINICAL INFECTIONS
■ Non-pregnant adults
– Bacteremia without focus or skin/soft tissue infections
■ Lead to endocarditis
– skin/soft tissue infections
■ cellulitis, abscesses, foot infection, or decubitus ulcers (DM)
– Septic Arthritis
– UTI
Streptococcus agalactiae

CLINICAL INFECTIONS
■ Elderly adults
– among persons ages 65 or older
– bacteremia, urinary tract infection, skin infection, and
pneumonia
■ Pneumonia
– Associated with dementia, neurological disease, and
tracheoesophageal fistulae
Streptococcus agalactiae

LABORATORY DIAGNOSIS
■ Specimen
– HVS, rectal swab, blood, skin/wound swab, urine, sputum
■ Specimen Work-up:
– Screening: (vaginal and rectal swab)
■ 3rd trimester (35th-37th week)
■ Lim Broth, TransVag Broth, or StrepB Carrot Broth
■ incubated at 35° C for 18 to 24 hours before being subcultured to SBA
– Non-screening – depends on the specimen received
Streptococcus agalactiae

LABORATORY DIAGNOSIS
■ Screening for asymptomatic bacteriuria
– GBS is reported when 104/mL is isolated in either pure
culture or mixed with a second microorganism
Streptococcus agalactiae

LABORATORY DIAGNOSIS
■ Microscopic: Gram-positive cocci
– short chains in clinical specimens
– longer chains in culture
■ Colony Morphology:
– on SBA: grayish white mucoid
colonies surrounded by a small zone
of β-hemolysis
Streptococcus agalactiae

LABORATORY DIAGNOSIS
■ Presumptive
– Hippurate hydrolysis
– CAMP test
– Bacitracin
■ Definitive
– Serologic: agglutination with specific
anti–group B antisera
Streptococcus agalactiae

■ Susceptibility testing and treatment

– All isolates are penicillin susceptible
– Susceptibility testing not appropriate in almost
all settings
– Exceptions are penicillin-allergic patients
Streptococcus agalactiae

■ Prophylactic Antimicrobial Therapy

– Women who are positive in screening

– who had a urine culture positive for GBS anytime during the
pregnancy

– A previous history of an infant with GBS disease

– Unknown GBS status at time of delivery (culture was not done)

Streptococcus agalactiae Review

■ Microscopic morphology
■ Colony Morphology
■ Culture requirements
■ Identification
■ Clinical infections
STREPTOCOCC
US
PNEUMONIAE
Streptococcus pneumoniae
■ lancet-shaped, gram-positive, facultative anaerobic bacteria with 100
known serotypes
■ cell wall contains an antigen, referred to as C substance (No
Lancefield grouping)
■ Infects human exclusively, no reservoir found in nature
■ Transmission:
– Direct person-to-person contact via respiratory droplets
– Autoinoculation in persons carrying the bacteria in their upper
respiratory tract
■ Carrier rate – 20%-40% in normal human nasopharynx (important
precursor to Pneumococcal disease)
Streptococcus pneumoniae
■ Pathogenesis and virulence
– Infection occurs after the colonization of the oropharynx and nasopharynx
– Inhalation causes the infection of the lower airways
– Capsular Polysaccharide
■ Plays a major role in the pathogenesis and virulence
■ escaping phagocytosis
– IgA1 protease
■ interferes with host defense at mucosal surfaces
– Neuraminidase
■ Removes sialic acid (protective barrier) on epithelial cells
– Pili
■ adherence of the organism to cellular surfaces and play a role in host
inflammation.
– Other Proteins:
■ pneumolysin, pneumococcal surface protein A, and autolysin
Streptococcus pneumoniae
■ Clinical Infections
– Pneumococcal pneumonia
– Otitis media
– Sinusitis
– Meningitis
– Bacteremia
– Septicemia
Streptococcus pneumoniae
■ Clinical Infections
– Pneumococcal pneumonia
■ an infection of the lungs that can cause mild to severe
illness in people of all ages
■ S/S: fever and chills, cough, rapid breathing or difficulty
breathing, chest pain
■ Complications: empyema, pericarditis, endobronchial
obstruction, atelectasis, pus formation
■ Pneumococcal pneumonia kills about 1 in 20 who get it
Streptococcus pneumoniae
■ Clinical Infections
– Otitis media
■ infection in the middle ear
■ S/S: ear pain; red, swollen ear drum; fever; sleepiness
■ Common form of infection; usually mild
– Sinusitis
■ an inflammation of the sinuses that can cause them to get
blocked and filled with fluid
■ S/S: headache; stuffy or runny nose; loss of the sense of smell;
facial pain or pressure and postnasal drip (mucus building up
in the back of the throat or nose)
■ Complications are rare, but include infection of the tissue
surrounding the eyes, bone infection, and a painful collection of
pus (abscess).
Streptococcus pneumoniae
■ Clinical Infections
– Bacteremia
■ Blood infection
■ S/S: fever, chills,low alertness
■ can lead to loss of limb(s).
– Septicemia
■ the body’s extreme response to an infection
– infection triggers a chain reaction throughout your body
■ a life-threatening medical emergency
– rapidly lead to tissue damage, organ failure, and death
■ S/S:
– Confusion or disorientation; shortness of breath; high heart
rate; fever, shivering, or feeling very cold; extreme pain or
discomfort; clammy or sweaty skin
Streptococcus pneumoniae
■ Clinical Infections
– Meningitis
■ an inflammation (swelling) of the
protective membranes covering
the brain and spinal cord
■ S/S: stiff neck; fever; headache;
photophobia (eyes being more
sensitive to light); confusion
■ those who survive may have
long-term problems, such as
hearing loss or developmental
delay
Streptococcus
pneumoniae
Laboratory diagnosis
■ Specimen:
– Blood, sputum, ear swab
■ Specimen Work-up:
– BHI or TSA with 5% sheep blood
– Chocolate Agar
– Add Optochin disc
– Incubate in an increased CO2
Streptococcus
pneumoniae
Laboratory diagnosis
■ Growth Work-up
– Gram Stain: GPC in pairs, lancet shape
– Colony:
■ round and usually wet, glistening and
mucoid
■ Alpha-hemolysis (partial,green)
– Optochin (diagnostic disc “P”)
susceptible
– Bile Solubility Test
STREPTOCOCCUS PNEUMONIAE
STREPTOCOCCUS PNEUMONIAE
Streptococcus pneumoniae
Streptococcus pneumoniae

Susceptibility testing and treatment

■ Penicillin remains the drug of choice
■ Penicillin-resistant isolates
– treated with Erythromycin or Chloramphenicol
■ Multidrug-resistant S. pneumoniae
– Resistant to two or more classes of antimicrobial agents:
■ erythromycin, tetracycline, chloramphenicol, and
trimethoprimsulfamethoxazole
 VIRIDANS
STREPTOCOCCI
 Viridans Streptococci

■ Normal flora ■ Classification:

– Upper respiratory tract
– S. mitis group
– female genital tract
– gastrointestinal tract. – S. mutans group
■ term viridans means “green,” – S. salivarius group
referring to the α-hemolysis – S. bovis group
many species exhibit
– Some are beta and non- – S. anginosus group
hemolytic – Pyogenic Group
■ Now termed as Oral
Streptococci
 Viridans Streptococci

General Characteristics:
■ spherical or ovoid cells that form chains or pairs
■ fastidious and mostly facultative anaerobic
■ non-motile
■ non–spore-forming
■ ferment carbohydrates with production of acid but not gas
 Viridans Streptococci

■ Pathogenesis and virulence

– virulence is low, BUT cause disease if host defenses are
compromised
– Virulence not well established
– Some produce:
■ Polysaccharide capsule
■ Cytolysin
■ Extracellular dextran and adhesins
■ M proteins
■ Extracellular enzymes such as SLO, hyaluronidase, and Dnase
 Viridans Streptococci

■ Clinical significance:
– Subacute bacterial endocarditis
– Gingivitis and dental caries (cavities)
– Others: meningitis, abscesses, osteomyelitis, and
empyema
 Viridans Streptococci

Laboratory Diagnosis
■ Specimen:
– blood, CSF, swab from affected tissue
■ Specimen Work-up
– SBA or CHOC (enriched agar or broth)
– Incubate in increased CO2 or anaerobic
conditions
 Viridans Streptococci

Laboratory Diagnosis
■ Gram Stain
– GPC in chains or pairs
■ Colony
– usually small colonies surrounded by
zone of alpha hemolysis
– vary in size and appearance
– some are beta or non hemolytic
Viridans Streptococci

Laboratory Diagnosis
■ Identification
– Species identification difficult
 Viridans Streptococci

Laboratory Diagnosis
■ Identification
– Differentiate from S.
pneumoniae:
■ Optochin resistance
– Less than 14mm
zone
■ Bile solubility
negative
 Viridans Streptococci

Laboratory Diagnosis
■ Identification
– Differentiate from
Enterococcus
■ Bile Esculin Test
■ Growth in 6.5% NaCl
 Viridans Streptococci

■ Susceptibility
– True cause of disease should be established before testing;
otherwise not appropriate
– viridans streptococci infections are treated with
penicillin
Review of Learning Outcomes

■ Compare the general characteristics and isolation of streptococci and similar

organisms.

■ Explain the Lancefield classification of streptococci.

■ Apply the knowledge of hemolytic patterns on sheep blood agar in the

identification of streptococcal isolates..

■ Describe the virulence factors and clinical infections associated with Streptococci

■ Discuss how to identify Streptococci from a clinical sample

THANK YOU