!!

Therefore these two groups are impossible to

Bacteriology: Gram- differentiate from one another with only microscopic
examinations
Positive Cocci ◼ Coagulase Test
Staphylococci • For initial differentiation of staphylococcal
species
• From the Greek word “staphule” – bunches of • Reagent: Plasma (usually rabbit plasma)
grapes, and “kokos” – berries. • Coagulase enzyme as the number one
• Gram positive cocci belonging to the virulence factor of S. aureus
Staphylococcaceae family • Positive result: clot formation/gel-like
• Spherical cells that appear in clusters • Coagulase-producing staphylococci:
• One of the most common isolates in the - S. aureus – most clinically significant
clinical microbiology laboratory; mostly part of - S. intermedius
the normal flora (skin, mucous membranes, - S. delphini
intestines). - S. lugdunensis and S. schleiferi –
• Catalase-positive; facultative except S. occasional due to clumping factor
saccharolyticus (obligate anaerobe

!! catalase (+) = presence of an enzyme that can degrade

hydrogen peroxide

• Non-motile, non-spore forming, glucose

fermenter !! the clumping factor present in S. lugdunensis and S.
• Colonies appear creamy, white or light gold, or schleiferi causes agglutination to the plasms
“buttery looking”, other species may have gray • Clinically significant coagulase-negative
colonies; others are β-hemolytic staphylococci:
• Pathogenic species: - S. epidermidis – associated with various
- S. aureus nosocomial infections
- S. epidermidis - S. saphrophyticus – associated with UTI in
- S. saprophyticus adolescent girls and young women
- S. lugdunensis - S. haemolyticus and S. lugdunensis –
- S. haemolyticus occasional isolates
Staphylococci vs. Micrococci Staphylococcus aureus

• True coagulase positive and the most clinically

significant staphylococci
• Golden yellow, and β-hemolytic colonies in
BAP
• Halophilic – can be cultured by adding 7/5% to
10% NaCl

Skin Infections
• Rothia mucilaginosa
• Aerococcus • Folliculitis
• Aliococcus - mild inflammation of hair follicles or
sebaceous glands
!! these are usually recovered from a human middle ear
- may be suppurative in nature
fluid
• Furuncles (boils)
• Micrococcus kristinae - Extension of folliculitis; large, raised
• Micrococcus varians superficial abscesses
• Carbuncles
!! positive for anaerobic acid production from glucose - Occurs when large, more invasive lesions
develop from multiple furuncles; can
 progress into deeper tissues and may - Promotes the formation of a fibrin layer
present with fever and chills around the abscess thereby protecting the
- May initiate inflammatory responses bacteria from phagocytosis
• Bullous impetigo ➢ Two types:
- Superficial infection commonly seen in 1. Cell-bound coagulase/clumping
children; characterized by crusty lesions factor (detected by coagulase test
and vesicles surrounded by a red border slide method)
2. Free coagulase (causes fibrin
Scalded Skin Syndrome
formation when bacterial cells are
• Bullous exfoliative dermatitis that occurs incubated in the plasma and
primarily in newborns and previously healthy detected by coagulase test tube
children but can be resolved within 4 days method)
• Caused by exfoliative/epidermolytic toxin • Hyaluronidase
• Adult cases were associated with chronic renal - Spreading factor; enhances invasion and
failure and compromised immunity survival in the host tissues
• Ritter Disease - Breaks down hyaluronic acid in
- Generalized condition where lesions cover intracellular ground substance of
approximately 90% of the body connective tissues
• Staphylokinase
Toxic Shock Syndrome - Dissolves fibrin clots
• Rare but potentially fatal, multisystem disease • Lipase
characterized by sudden onset of fever, chills, - Reason why it can survive in an oily
vomiting, diarrhea, muscle aches, and rashes envirnment
• Can quickly progress to hypotension and shock - Produced by coagulase-positive and
coagulase staphylococci
• First described by Todd (1978) and was
- Essential for the survival in sebaceous
associated with tampon use
areas of the body
• Caused by toxic shock syndrome toxin 1 (TSST-
- Involved in skin infections
1)
• Deoxyribonuclease (DNAse) and phosphate
Food Poisoning - Decreases exudate viscosity, allowing the
bacteria to be mobile
• Caused by ingestion of enterotoxins
- Destroys DNA
(commonly by enterotoxin A) formed outside
• Β-lactamase
the body.
- Breaks down the β-lactam rings and
• Occurs when food Is contaminated with
related drugs
enterotoxin-producing S. aureus by improper
- More than 90% of clinical isolates of
food preparation (contamination) and
staphylococci are penicillin-resistant due
storage.
to this enzyme
!! In Philippines, the most incriminated food that can be • Heat-stable enterotoxin
most likely to be infected by S. aureus is spaghetti (as it - Induces vomiting by stimulation of vagus
provides all the necessary nutrient for the bacteria) nerve
- Stable at 100°C for 30 minutes and
Other associated conditions resistant to enzymatic hydrolysis
• Septic arthritis - Examples: enterotoxin A, B, C1, C2, D, E, G
• Osteomyelitis to J (A, B, and D are commonly implicated
• Urinary tract infection with food poisoning; B is associated with
• Acute bacterial endocarditis pseudomembranous enterocolitis
• Bacteremia and sepsis • Panton-Valentine Leukocidin (PVL)
◼ Virulence factors - Pore-forming toxin that destroys WBCs
• Coagulase (neutrophils and macrophages).
- Converts fibrinogen to fibrin - Responsible for necrotizing skin and soft
tissue infections
 • Hemolysin !! MRSA infection is costly and post a serious threat to the
- Cytolytic toxin that destroys RBCs health institution
➢ Four types:
➢ Three types:
1. Alpha (α) - predominant toxin;
1. Hospital-acquired
also destroys platelets and
2. Community-acquired
macrophages; causes severe
3. Healthcare-associated
tissue damage
community-onset
2. Beta (β) – also destroys
• CHROM agar – MRSA produce mauve-colored
sphingomyelin; has enhanced
colonies upon hydrolysis of chromogenic
activity upon incubation at 35°C
substrate
and subsequent exposure to 4°C
• Gold standard test: detection of mec A gene by
3. Delta (δ) – associated with PVL
molecular methods
4. Gamma (γ) – less toxic compared
to α and β; produces by all S.
aureus strains and causes
edematous lesions
• Epidermolytic toxin A and B
- Serine protease that splits the intracellular
bridges of epidermis and causes extensive
sloughing, producing burn-like lesions
- Destroys the stratum granulosum !! mec A gene = it produces altered trans-peptidase
• Toxic shock syndrome toxin 1 (TSST-1) enzymes in the cell wall of the S. aureus making the action
- Chromosomal-mediated toxin penicillinase resistant penicillin like nafcillin, oxacillin
- Stimulates the production of large ineffective
amounts of cytokines Coagulase-negative Staphylococci
• Protein A
- Immunogenic molecule found on the
Staphylococcus epidermidis
bacterial cell wall
- Exerts antiphagocytic effects by binding • Indigenous microbiota of the skin
the Fc portion of IgG and other opsonin • Contaminant of medical instruments,
catheter, CSF shunts, and implanted medical
!! not all strains of S. aureus have or execute all of these
devices (e.g. prosthetic heart valves)
factors
• Characteristics on BA: white, opaque, pinhead,
Methicillin-resistant S. aureus (MRSA) gamma-hemolytic
• Coagulase-negative; no growth on MSA;
• Strain of S. aureus that is resistant to
susceptible novobiocin (5µg)
antibiotics such methicillin, nafcillin, and
oxacillin Staphylococcus saprophyticus
• Can be acquired after prolonged stay in the
• Commonly associated with community-
hospital, close contact with carriers, or after-
acquired UTI among young and sexually-active
effects of broad-spectrum antibiotic
females
treatment
• adheres more effectively to the epithelial
• Controlled by proper isolation of the
lining of urogenital tract than other CoNS
organism, rapid identification, handwashing,
treatment of carriers, and strict compliance of • characteristics on BA: white, opaque, slightly
infection control programs larger than pinheads, gamma-hemolytic; some
may produce yellow pigments
• colony count of <10,000 CFU/mL in urine
culture is still considered significant

Staphylococcus lugdunensis

• occasionally positive for coagulase test slide

method but negative for tube method
 • can be more virulent than other CoNS and may !! test organism should not be taken from BAP due to the
mimic S. aureus infections peroxidase-like activity of the hemoglobin that can cause
• contains mc A gene for oxacillin resistance false positive
• related diseases include endocarditis,
◼ Coagulase test
meningitis, septicemia, UTI, and skin and soft
• Best single criterion of S. aureus pathogenicity
tissue infections
• Reagent: rabbit plasma
!! some strains may have clumping factors and can be • Anticoagulant: EDTA
mistaken as S. aureus if only slide method is used • Use of citrated plasma may yield false-positive
results due to citrate-utilizing organisms
Resistant Genes by Staphylococci
• Slide method
◼ Erythromycin ribosomal methylase (Erm) gene - For screening of catalase-positive cocci
• Enzyme-inactivating gene - Detects cell-bound coagulase (clumping
• Codes for the methylation of 23s rRNA, factor)
resulting in the resistance to erythromycin and - Positive result: clot formation within 30
either conducible or constitutive resistance to seconds
clindamycin - Other cell-bound coagulase-positive
◼ Methionine sulfoxide reductase (msr) A gene staphylococci:
• Code for an efflux mechanism which results in ➢ S. lugdunensis
the resistance to erythromycin but ➢ S. schleiferi
susceptibility to clindamycin • Tube methods
- Detects unbound/free coagulase
Laboratory Diagnosis for Staphylococci - Plasma in test tube is inoculated with test
◼ Gram stain organism and incubated at 35°C
• G(+) spherical cells that appear singly, in pairs, - Positive result: clot formation after 1-4
or in clusters hours of incubation
◼ Culture - Other free coagulase-positive
• Grows easily on 5% sheep BA and CA staphylococci:
• Selective media (e.g. MSA, Columbia CNA, ➢ S. hyicus
PEA) may be used for heavily contaminated ➢ S. intermedius
specimens ➢ S. lutrae
• CHROMagar – selective and differential ➢ S. delphini
medium for the isolation MRSA ➢ S. schleiferi subsp. coagulans

!! Columbia CNA and PEA are particularly selective for G (+)

bacteria

◼ Catalase test
• A heme enzyme that catalyzes the
decomposition of H2O2 to H2O and O2
• Differentiates staphylococci from streptococci
• Reagent: 3% H2O2
• Positive result: effervescence

◼ Mannitol fermentation
• Used for differentiating pathogenic form non-
pathogenic staphylococci
• Culture medium: MSA (pH indicator: phenol
red
• Pathogenic staphylococci ferments mannitol
with subsequent production of an acid
 • Positive result: yellow S. aureus colonies - Cefoxitin isused to induce the expression
surrounded by yellow halos of PBP2a in mecA-containing strains of
◼ Pyrrolidonyl arylamidase (PYR) test staphylococci
• Differentiates coagulase-positive • Double-disk diffusion test (D-test)
staphylococci in slide method by detecting - Used for detecting inducible resistance to
arylamidase-producing staphylococci clindamycin
• Reagent: L-pyrrolidonyl-β-naphthylamide and - An erythromycin (15µg) and clindamycin
p-dimethylaminocinnamaldehyde (2 µg) are placed 15-26 mm apart on MHA
• Positive result: cherry-red color or BA with the test organism incubated at
• S. lugdunensis and S. schleiferi are PYR positive 35°C for 18-24 hours
- Positive result: flattening of the
clindamycin zone near erythromycin (D-
zone)
◼ VP test
• Differentiating S. aureus and S. intermedius
• S. aureus produces acetoin (acetylmethyl
carbinol) from glucose
◼ Tellurite glycine agar • positive result: pink to red color at the surface
• Used for the quantitative detection of of MRVP medium
coagulase-positive staphylococci
• Positive result: jet-black colonies of S. aureus

◼ DNAse test
• Used for identification of pathogenic
staphylococci that produce DNAse
• Culture medium: DNA-methyl green agar
◼ AST • Test organism is inoculated onto the medium
and incubated at 35°C to 37°C for 18-24 hours
• Oxacillin-screen plate
- Used to screen MRSA in clinical specimens • Positive result: clear zone around the colonies
- MHA with 4% NaCl and 6 µg/mL oxacillin • S. aureus produces positive result
- Test organism is spot-inoculated using
cotton swab and incubated at 35°C for 24
hours
- Advantageous in detecting BORSA in
clinical samples
- Unreliable in detecting oxacillin-resistant
CoNS
• Vancomycin-screen plate ◼ Β-lactamase test
- S. aureus isolates must be screened with
• Cephalosporinase Test
6µg/mL vancomycin incorporated into BHI
- Most useful and widely used
medium
- Reagent: cephalosporin or cefinase disk
- Any growth is considered significant with nitrocefin (substrate)
• Cefoxitin disk diffusion (30 µg) - Test organism is applied directly on the
- Preferred method for detecting oxacillin pre-moistened disk
resistance in both S. aureus and S. - Positive result: deep pink or red color in 60
lugdunensis minutes
- Improves detection of MRSA
 • Acidimetric method - Clear zone of hemolysis around the colony
- Reagent: citrate-buffered penicillin
- pH indicator: phenol red
- positive result: red to yellow color change
• iodometric methods
- reagents: phosphate-buffered penicillin
and starch-iodine complex
- negative result: purple-colored solution
- positive result: colorless solution
◼ Latex agglutination
• Used for detecting clumping factor and - Examples: S. pyogenes and S. agalactiae
protein a • Alpha hemolysis
• Useful for detecting altered penicillin-binding - Exhibit partial/incomplete hemolysis of
protein (PBP), and oxacillin-resistant strains red blood cells around the colonies
• It can be used for both S. aureus and CoNS - Greenish discoloration around the
◼ Polymyxin Sensitivity test colonies
• S. aureus is resistant to polymyxin - Examples: S. pneumoniae
• Gamma hemolysis
- Non-hemolytic
- Colonies are surrounded by intact red
blood cells
- Example: S. bovis

◼ Lancefield Classification
Streptococci and Enterococci (Last Page)
• Belong to the Streptococcoeae family Group A Streptococci
• Gram-positive, Catalase negative cocci,
arranged in pairs • Not considered a part of the normal
• Some species are part of the normal microbiota (overt pathogen)
microbiota but can cause life-threatening • Transmitted via sneezing or coughing (droplet
infections to normally sterile body sites transmission)
• Mostly facultative anaerobes (except • Resistant to drying and can be recovered from
Peptostreptococcu – obligate anaerobe); some swab several hours post-collection
are aerotolerant anaerobes and capnophilic
• Growth is enhanced by blood, serum, or
glucose added in the medium
• All streptococci (except S. pneumoniae and
viridans group) are included in the Lancfield
classification
• Colonies are gray, pinpoint, and translucent to
slightly opaque; some are mucoid
• Basic characteristics: negative for catalase, Streptococcus pyogenes
oxidase, and gas production; non-motile, CHO
• Most clinically significant
fermenter
• “Fever-producing” and “flesh-eating”
Classification of Streptococci bacterium
• Colonies on BA: small translucent, smooth,
◼ Smith and Brown Classification
and exhibit well-defined β-hemolysis
• Beta hemolysis
- Exhibit complete lysis of red blood cells
around the colony
◼ Virulence Factors
• M protein
- Primary virulence factor
- Resists phagocytosis and aids in mucosal
cell adherence
- Attached to the peptidoglycan
• F protein
- Mediates epithelial cell attachment
• Lipoteichoic acid
- For adherence to respiratory epithelium
• Hyaluronic acid capsule
- Weakly immunogenic - Solubilizes the ground substance of host
- Prevents opsonin-mediated phagocytosis connective tissue
and masks the cell’s antigen • Streptococcal pyrogenic exotoxins
• Streptolysin O - Formerly called erythrogenic toxin
- Oxygen-labile hemolysin - Four immunologically distinct types in S.
- Responsible for sub-surface hemolysis on pyogenes: SpeA, SpeB, SpeC, and SpeF
BA that is incubated anaerobically - Highly mitogenic proteins (superantigens)
(demonstrated by “stabbing” the agar to - SpeB mediates the rashes seen in scarlet
create anaerobioses and observed sub- fever
surface hemolysis) Bacterial Pharyngitis
- Anti-streptolysin O (ASO) – antibody
produced form recent infection with S. • Most common clinical manifestation of GAS
pyogenes infection (also tonsillitis)
• “Strepto throat” is often seen in children
between 5-16 years old
• Signs and symptoms: sore throat, malaise,
fever, headache, nausea, vomiting, abdominal
pain, inflamed tonsils, and pharynx, swollen
cervical lymph nodes
• Incubation is 1-4 days; symptoms subside 3-5
days unless complication occur
• Spread via droplet transmission and close
contact
• Diagnosis relies on throat swab cultures or
antigen detection
• Highly virulent strains can cause outbreaks of
sore throat and scarlet fever in schools and
camps
• Streptolysin S
- Oxygen-stable hemolysin
- Responsible for surface hemolysis on BA
that is incubated aerobically
- Non-immunogenic
• DNAse
• Streptokinase
- Dissolves fibrin clot
- Binds to plasminogen and activates it to
plasmin
• Hyaluronidase
- Spreading factor
Pyodermal Infections ◼ Scarlet fever
- Caused by pyrogenic exotoxins
◼ Impetigo
- Characterize by punctate exanthem
- Localized skin disease that begins as a
overlying a diffuse erythema appearing
small vesicle that progresses to weeping
initially on the neck and upper chest 1-2
lesions; usually seen in young children (2-
days after infection
5 years old)
- Acquired through abrasion or insect bite
- Also known as galloping gangrene or flesh-
eating bacteria syndrome and may lead to
necrotizing fasciitis

- Rash spreads to the trunk and extremities;

disappears over the next 5-7 days
followed by desquamation
- Communicable and spread via droplet
transmission
◼ Erysipelas - “Strawberry tongue”
- Rare infection of the skin and
subcutaneous tissues usually seen in
elderly patients
- Characterized by an acute spreading skin
lesion that is intensely erythematous with
a plainly demarked but irregular edge

• Dick Test
- Aka erythrogenic toxin test
- Susceptibility test
- Positive result: skin redness
• Schultz-Charlton Test
◼ Cellulitis - Aka anti-erythrogenic toxin test
- Can develop following deeper bacterial - Diagnostic test
invasion - Positive result: “blanching phenomenon”
- It can be serious of life-threatening with (fading rash)
bacteremia or sepsis Necrotizing Fasciitis
- It may lead to gangrene in patients with
peripheral vascular disease or diabetes • An invasive infection characterized by rapidly
progressing inflammation and necrosis of the
skin, subcutaneous fat, and fascia
• Uncommon but life-threatening (mortality for
untreated infection = >70%)
• Types:
➢ Type 1 NF – polymicrobial cause
(aerobic and anaerobic)

!! Salt-water NF caused by Vibrio spp. e.g. V.

parahemolyticus and V. vulnificus

➢ Type 2 NF – GAS
 ➢ Type 3 NF – clostridial myonecrosis - Group C is susceptible; Group G is variable
(“gas gangrene”)

• Sulfamethoxazole and Trimethoprim Test (SXT

Test)
- Positive result: resistance
Streptococcal Toxic Shock Syndrome - Groups A and B streptococci are resistant
• A condition in which the entire organ system to SMZ
collapses, leading to death - Other β-hemolytic streptococci, viridans
streptococci, and S. pneumoniae are
• Exact portal of infection is unknown but has
susceptible
been implicated with minor injuries and
surgeries
• Initial infection is severe (pharyngitis,
peritonitis, cellulitis, wound infection)
• SPE-producing GAS (specifically SpeA) plays
major role in the infection

Post-streptococcal Sequelae

◼ Rheumatic Fever
• Typically follows strep throat
• Characterized by fever and inflammation of
the heart, joints, blood vessels, and
• Pyrrolidonyl Arylamidase (PYR) Test
subcutaneous tissues
- Detects the presence of the PYR enzyme in
• Chronis and progressive damage to the heart
streptococci
valves -> most serious result
- S. pyogenes is the only β-hemolytic
• Repeated infections can lead to further valve
Streptococcus that tests positive for PYR
damage (rheumatic heart disease)
test
◼ Acute Glomerulonephritis
- Positive result: cherry red solution
• Aka Bright’s disease
- Enterococci also yields positive result for
• Usually follows cutaneous or pharyngeal
this test
infections
• Circulating immune complexes are deposited Serological Tests
in the glomeruli, and subsequent
• Anti-streptolysin O
inflammatory responses cause damage to the
• Anti-DNAse
kidneys and impaired glomerular function
• Anti-streptokinase
Laboratory diagnosis • Anti-hyaluronidase
◼ AST Group B Streptococci
• Bacitracin/Taxo A Test (0.04 units)
- Differentiates S. pyogenes from other β- • Part of the normal flora of the female genital
hemolytic streptococci tract and lower GI tract
- Positive result: susceptible (any zone of • Transmitted through contaminated hands
inhibition) • May cause premature membrane rupture and
- Helpful in identifying GAS in throat may infect the baby during childbirth
cultures
Streptococcus agalactiae ◼ Hippurate Hydrolysis Test
• Used to differentiate S. agalactiae and other
• The only species in group B classification
β-hemolytic streptococci
• Sialic acid-containing polysaccharide capsules
• Detects the presence of Hippurate hydrolase
-> virulence factor
in S. agalactiae
• Other factors include:
• Reagent: sodium Hippurate and ninhydrin
- CAMP factor
• Positive result: purple solution after addition
- Hemolysin
of ninhydrin
- Neuraminidase
- DNAse
- Hyaluronidase
- Protease
• colonies in BA appear grayish-white and
mucoid with a small zone of β-hemolysis
• US CDC recommends that all pregnant women
should be screened for GBS at 35-37 weeks of
pregnancy
◼ Serotyping
• Coagglutination – use of bacterial cells

Disease caused by GBS infection

• Latex agglutination – use of latex
• Neonatal meningitis and sepsis
• Pneumonia
• Postpartum infection
• Osteomyelitis
• UTI
• Endocarditis

Laboratory Diagnosis

◼ Christie-Atkins-Munch-Petersen (CAMP) Test

• Used to differentiate S. agalactiae from other Culture media for GAS/GBS Isolation
β-hemolytic streptococci
• A β-lysin strip or β-lysin-producing strain of S. • BA, CA, Columbia CNA, PEA, Carrot broth, Lim
aureus is inoculated on BA broth, Granada agar
• Incubated in an aerobic condition at 35°C • CA is incubated with 5% to 10% CO2
• Positive result: arrowhead- or bowtie- shape • Carrot broth and Granada agar are both
β-hemolysis near S. aureus growth selective and are used for vaginal and rectal
swabs for pregnant women
◼ BA with SXT
- for cultivating GAS from throat swabs;
suppresses growth of normal microbiota
◼ Todd-Hewitt broth (transport) with 10 µg/mL
colistin and 15 µg/mL of nalidixic acid
- for detecting GBS in vaginal or rectal
swabs
◼ Carrot broth
 - chromogenic medium; will change from • S. mitis group – common cause of SBE (Sub-
colorless to yellow or orange in the acute Bacterial Endocarditis)
presence of GBS • S. bovis group – have group D antigens on their
◼ Granada agar cell walls (similar with enterococci)
- yellow to orange colonies of GBS • S. gallolyticus subsp. gallolyticus – often
◼ Lim broth isolated in the blood of GI carcinoma patients
- improves recovery of GBS • S. anginosus group – species may have
Lancefield group A, C, F, G, or N antigens or
Group C and G Streptococci
none
• Recovered from the upper respiratory tract,
Laboratory Diagnosis
vaginal, and skin
• Also possess M protein ◼ Leucine aminopeptidase test
• Group c streptococci are animal pathogens • LAP hydrolyzes peptide bonds that are
and serve as the main source of streptokinase adjacent to free amino acids
• Reagents:
!! streptokinase is isolated for its therapeutic actions as a
1. Leucine-β-naphthylamide
thrombolytic agent in patients with myocardial infarction
2. p-dimethylaminocinnamaldehyde
and stroke
• End product: β-naphthylamine
• Examples: S. dysagalactiae subsp. equisimilis • Positive result: red color
and S. equi subsp. zooepidemicus ➢ Positive: viridans, enterococci, GAS,
GBS, S. pneumoniae
Viridans Streptococci
➢ Negative: Aerococcus and
• Not included in the Lancfield classification Leuconostoc
• Constituents of normal flora of the upper ◼ Vouges Proskauer test
respiratory tract, female genital tract, and GI • Detects production of acetoin/acetylmethyl
tract carbinol from glucose fermentation
• Viridans – “green”; α-hemolytic streptococci • Culture medium: VP broth
(some are β- and γhemolytic); lacks Lancefield • Reagents:
group antigens - α-naphthol
• Virulence factors: polysaccharide capsule, - KOH
cytolysin, extracellular dextran, and adhesin • Positive result: pink to red color at the surface
• Diseases: subacute bacterial endocarditis, of VP medium
gingivitis, dental caries, osteomyelitis, • S. anginosus, S. bovis, and S. mutans group
meningitis yield positive results in VP test
◼ β-D-Glucuronidase test
• Enzyme that hydrolyzes β-D-glucopyranosid-
uronic derivatives to aglycons and D-
glucuronic acid
• Reagents:
➢ 4-methylumbelliferyl-β-D-glucuronide
(MUG)
• End product: 4-methylumbelluferyl (4-MU)
• Positive reaction: blue fluorescence
• Large colony forming β-hemolytic groups C
Groups of Viridans Streptococci and G yields positive results
◼ AST
• Group C streptococci are susceptible to
bacitracin and SXT
• Group G streptococci may be susceptible or
• S. mutans group – most isolated viridans
resistant to bacitracin
streptococci; cariogenic, sucrose fermenting
Laboratory diagnosis of S. bovis Group (GDS) Diseases caused by Pneumococcal Infection

◼ Growth on bile esculin medium ◼ Lobar pneumonia

• Reagents: esculin and 1% to 4% bile salt • Characterized by the presence of high volume
• Positive result: black complexes in the agar of fluids in the alveoli, which further spreads
within 48 hours the bacteria throughout the lungs
• 6.5% NaCl (nutrient broth base) test – no • Sputum microscopy shows presence of large
growth / no turbidity number pneumococci and WBCs
• PYR test – no color change ◼ Otitis media
• Penicillin test – susceptible / sensitive ◼ Meningitis
◼ Sepsis
◼ Endocarditis and peritonitis
◼ Atypical HUS

Laboratory Diagnosis

• Specimens: sputum, blood, CSF

• Gram stain:
- Oval or lancet-shaped cells in pairs of
short chains
- WBCs and Gram-positive diplococci in CSF
Streptococcus pneumoniae direct microscopy
• Culture
• Also known as pneumococcus
- Young colonies: “dome-shaped”,
• A more virulent member of the S. mitis group;
glistening, wet, and mucoid
contains C polysaccharide (similar with
- Old colonies: “coin with raised rim” or
Lancefield classification)
“dimple-shaped”
• Reacts with human CRP and form precipitates
• Optochin/Taxo P Test
• Most common cause of bacterial pneumonia - 6 µm or 10 µm
among elderly and immunocompromised optochin/ethylhydroxycuprein in BA
patients
- Susceptible: S. pneumoniae
• G (+) cocci in pairs in pairs; oval or lancet- - Resistant: other α-hemolytic streptococci
shape
• Bile Solubility Test
• Colonies in BA appear flat with depressed - Differentiates pneumococci and viridans
centers, mucoid, α-hemolytic streptococci
• Virulence factor: polysaccharide capsule - S. pneumoniae cells are lysed in the
• Other factors: hemolysin, IgA protease, presence of bile salts
neuraminidase, hyaluronidase • Neufeld-Quellung Test
- Capsular swelling test
- Capsule of S. pneumoniae swells in the
presence of specific anti-capsular serum
• Coagglutination Test
- Use of bacterial cell-bound antibody to
enhance visualization of antigen-antibody
reaction
• Francis Test
- Skin test to detect the presence of
antibodies against pneumococci

Enterococci

• Belong to the family Streptococcaceae and

formerly classified as group D streptococci
 • Normal microbiota of human and animal • Formerly known as nutritionally variant
intestinal tract streptococci
• Not highly pathogenic but frequently causes • Aka pyridoxal-dependent, thiol-dependent,
nosocomial infections; resistant to many and symbiotic streptococci
antibiotics • Normal microbiota of the mouth and GI tract
• Species: E. faecalis, E. faecium, E. avium, E. • Abiotrophia will not grow on BA or CA unless
gallinarum, E. durans, and E. raffinosus pyridoxal is added
• Culture appear as “satellites” around a
!! E. faecalis is the most common isolates
pyridoxal-producing organism
• Variable hemolytic patterns in BA • Microscopy: Gram-variable and pleomorphic
• Virulence factors: extracellular serine forms
proteases, surface adhesin proteins, • Growth factors: thiol compounds such as
gelatinase, and cytolysin pyridoxal and cysteine
• Related infections: UTI endocarditis,
bacteremia, and wound infection

Laboratory Diagnosis

◼ Gram staining
• G (+) cocci in pairs and long chains
◼ Culture
• TSB or BHI with 5% sheep’s blood
• Recommended media for contaminated
specimens: Streptococcus-like Organisms
➢ Bile esculin azide
◼ Gowth in bile esculin medium
Aerococcus
- Determines the ability of the organism to
grow in the presence of 4% bile and the • Common airborne bacterium
ability to hydrolyze esculin • Resembles viridans streptococci in culture but
- Reagents: esculin and 1-4% bile salt is microscopically similar with staphylococci
- Positive result: black complexes in the (appears in clusters or tetrads)
medium within 48 hours • Has weak catalase/pseudocatalase reaction
◼ 6.5% NaCl (nutrient broth) test and grows on 6.5% NaCl
- Enterococci grow on this medium while • Species:
non-enterococci (GDS) do not ➢ A. viridans – positive for BE and PYR
- Growth is indicated by turbidity ➢ A. urinae – negative for BE and PYR
◼ Acid production from CHO utilization
- Enterococci utilize methyl-α-D- Gemella
glucopyranoside (MGP), producing an acid • Has similar colony morphology with viridans
byproduct streptococci
◼ PYR test • G (+) cocci but are easily decolorized (appear
- All enterococci isolated from humans as G (-) cocci in pairs, tetrads, clusters, or short
produce PYR enzyme chains)
- Reagent: L-pyrrolidonyl-β-naphthylamine
• Have been isolated from cases of endocarditis,
and p-dimethylaminocinnamaldehyde
wounds, and abscesses
- Positive result: cherry-red color
• Species: G. haemolysan, G. morbillorum, G.
◼ Antimicrobial susceptibility
bergeriae, G. sanguinis
- Penicillin test – enterococci are resistant
- Efromycin acid disk (100 µg) – enterococci Lactococcus
are resistant
• G (+) cocci that occurs singly, in pairs, or in
Abiotrophia and rhelicatella chains
 • Physiologically similar with enterococcie;
previously classified as group N Streptococci
• Acid production from carbohydrates is useful
in differentiating Lactococcus from
enterococci
• Have been isolated in patients with UTI and
endocarditis

!! does not utilize CHO

Leuconostoc

• Have phenotypic and biochemical semblance

with Lactobacillus spp., viridans streptococci,
Pediococcus spp., and enterococci
• Frequently found on plant surfaces,
vegetables, and milk products
• Opportunistic pathogens in
immunocompromised patients or those
undergoing vancomycin treatment
• Grows on BEA and 6.5% NaCl; negative for LAP
and PYR
• Species:
➢ L. citreum
➢ L. cremoris
➢ L. dextranicum
➢ L. lactis
➢ L. mesenteroides
➢ L. pseudomesenteroides

Pediococcus

• Facultatively anaerobic, G (+) cocci arranged in

pairs, tetrads, or clusters; grows at 45°C
• Resistant to vancomycin and may be
misidentified as viridans streptococci
• Have been associated with patients with GI
disorders or those with previous abdominal
surgery; bacteremia and meningitis
• Positive for BE and LAP; negative for PYR