Center for Disease Control and

Prevention(CDC): Universal Precautions!

• use of personal protective equipment (PPE)
• Work practice controls should be strictly
implemented
HEPA FILTER
High-efficiency particulate air

• It has the ability to kill 99.9% of harmful air particles. These

particles are usually 0.3 microns or less..
• Class I and II Biosafety cabinets are used for Biosafety levels I
and II but, when used correctly in conjunction with useful
microbiological techniques, these provide an effective
containment system for safe manipulation of moderate and
high-risk microorganisms.
• Class III BSCs are most suitable for work with hazardous agents
that require Biosafety Level 3 or 4.
 Class I

• Operator and environment protection

• These enclosures are used to protect the user and laboratory
environment from Aerosol contamination.
Class II

• Operator, samples and environment protection

• 30% air exhaust, 70% air recirculation
• This type of biological safety cabinet not only protects operators,
but also protects the samples and laboratory environment.
Class III

• Having three High-Efficiency Particulate Air (HEPA) filters, the cabinet

ensures maximum safety for the user and the environment. The Class
III Biological Safety Cabinet has a complete and airtight sealed
chamber for working with hazardous materials.
• Examples:
• The size range of Escherichia coli bacteria is between
1.1 and 1.5 um in diameters.
• The size range of Bacillus anthrac is between 1.0 and
1.2um.
• Bacillus subtilis range between 0.25 and 1.0um in
diameter.
Bacteriology:

GRAM POSITIVE COCCI

A. FAMILY MICROCOCCACEAE
B. FAMILY STREPTOCOCCACEAE
 FAMILY MICROCOCCACEAE
General Characteristics
• Normal inhabitants of the skin and mucous membrane
• Gram + cocci, usually in clusters
• Catalase (+)

Genera:

1. Staphylococcus
2. Micrococcus
 FAMILY MICROCOCCACEAE

Staphylococcus Micrococcus

Coagulase Variable (-)

OF glucose Fermenter Oxidizer

Bacitracin Resistant Susceptible

Lysostatin Susceptable Resistant

Modified Oxidase (-) (+)

 FAMILY MICROCOCCACEAE
Clinically Significant Species
1. Staphylococcus aureus
Virulence factors
1. enterotoxins
a. enterotoxins A - E -intestinal tract toxin
-associated with food poisoning
- resistant to gastric juice
- heat stable
b. enterotoxin F/exotoxin C /TSST-1
-produced by S. aureus phage group 1
- causes toxic shock syndrome (TSS)
 FAMILY MICROCOCCACEAE
Clinically Significant Species
1. Staphylococcus aureus
Virulence factors
2. exfoliative toxin/epidermolytic toxin
- produced by S. aureus phage group II
-causes scalded skin syndrome (SSS)
3. Protein A
-binds the Fc portion of Ig
-aids the bacterium avoid phagocytosis by interfering with opsonization
4. cytolytic toxins- alpha, B, delta hemolysin , Panton-Valentine leukocidin
5. enzymes-coagulase, hyaluronidase (spreading factor), lipases
1. Staphylococcus aureus
Epidemiology

S. aureus inhabits the anterior

nares and perineum
1. Staphylococcus aureus
Clinical Infections
1. skin and wound –
Suppurative and pyogenic; abscess filled with pus

a. folliculitis - inflammation and infection of hair

follicles
b. boils /furuncles - lesions of folliculitis may
develop into a deeper inflammatory nodule
called a furuncle
c. Carbuncle-abscess that extends even more
deeply into the subcutaneous fat and may have
multiple draining sites
d. impetigo - pustules are larger and surrounded
by a small zone of erythema compared to strep
impetigo; highly contagious
1. Staphylococcus aureus
Clinical Manifestations

2. food poisoning
- caused by enterotoxins A- E; short incubation period
2-8 hours
- - food poisoning by enterotoxin B is more severe

3. TSS (toxic shock syndrome) caused by phage group

producing TSST-1
- multisystem diseáse characterized by high fever,
hypotension, shock and rash
- associated with tampon use but may also be seen in
males and non-menstruating females
1. Staphylococcus aureus
Clinical Manifestations
4. Scalded Skin Syndrome(SSS) / Ritter disease/ Lyell's
syndrome

- exfoliative dermatitis; exfoliative or epidermolytic

toxin; caused by phage group
- more common in newborns and adults with chronic
renal failure and impaired immune system toxic
epidermal neerolysis

5. Nosocomial Infections: bacteremia, endocarditis,

pneumonia

6. Septic arthritis in prepubertal children

 FAMILY MICROCOCCACEAE
Clinically Significant Species
2. Staphylococcus epidermidis
- associated with hospital-acquíred infections
- most common of the CoNS
- Slime enables the organism to adhere to the surface of prosthetic
devices
1. most common cause of hospital-acquired UTI
2. prosthetic valve endocarditis
3. septicemia in immunocompromised patients
 FAMILY MICROCOCCACEAE
Clinically Significant Species
3. Staphylococcus saprophyticus
associated with UTI among young sexually
active females
- adheres to the urogenital tract; rarely found
in other skin areas and mucous membranes
 FAMILY MICROCOCCACEAE
Laboratory Diagnosis
• 1. Microscopic Exam
• Appear Gram-positive cocci Singly, in pairs, or in clusters
(bunches of grapes)

2. Culture
Appear as cream or off-white colonies on blood agar(BA)
 FAMILY MICROCOCCACEAE
Laboratory Diagnosis
Mannitol salt agar (MSA)
- selective and differential medium useful in recovery and
identification of Staphylococcus aureus from specimens with mixed
flora

• high salt concentration of 7.5% inhibits most g(-) and g(+) bacteria except
staphylococçus species
• mannitol fermentation produces acid products and the lowered pH changes
the color of phenol red to yellow
• colonies of S. aureus appear yellow while S, epidermidis are reddish
 FAMILY MICROCOCCACEAE
Laboratory Diagnosis
Columbia colistin-nalidixic acid agar (CNA)
- selective enrichment medium for isolation of g(+) cocci –
- colistin and nalidixic acid +sheep's blood for enrichment

Phenylethyl alcohol agar (PEA)

- selective medium to isolate g(t) cocci from specimens with mixed
flora phenylethyl alcohol + sheep's blood
- phenylethyl alcohol inhibits facultative g()bacilli (esp) swarming
Proteus) but permits the growth of g(t)cocci
 FAMILY MICROCOCCACEAE
Laboratory Diagnosis
3. Biochemical Tests
• Catalase test
-used to differentiate staphylococci from streptococci
-drop of 3% H202 on a slide + organism
(+) Rxn: bubbles

• Modified Oxidase (Microdase)

-Used to differentiate Micrococcus from coag(-)staph
-tetramethyl-p-phenylenediamine dihydrochloride +
dimethyl sulfoxide (DMS0) positive reaction: purple in 30
secs
 FAMILY MICROCOCCACEAE
Laboratory Diagnosis
• Coagulase
-used to differentiate S. aureus from coag-negative staph
-major marker for S. aureus; coagulase clots plasma

2 types of coagulase enzymes:

1. cell-bound coagulase / clumping factor
- detected by the slide coagulate test; used to screen colonies that resemble S.
aureus
- on a glass slide make a suspension of organism with NSS then add a drop of plasma;
(+) Rxn: clumping
 FAMILY MICROCOCCACEAE
Laboratory Diagnosis

2. free coagulase / extracellular free coagulase

-detected by the tube coagulase test

-5% of S.aureus do not produce cell-bound coagulase
- negative slide coagulase test result must be confirmed with the tube method
- isolates that do not produce either bound or free coagulase are reported as
coagulase-neg staphylococcus in a tube: plasma + organism; incubate at 37 C for
4 hours the clot formed in the tube may havea tendency to autolyse becaus e of
fibrinolysin (staphylokinase)
(+) Rxn: clot
 FAMILY MICROCOCCACEAE
Laboratory Diagnosis
➢ latex agglutination (ex. Staphaurex) expensive
-utilize plasma-coated latex particles
-plasma attached to latex) detects clumping factor and protein A in the cell wall of S. aureus

• Novobiocin resistance

-urine isolates that are coagulase-negative are further tested to presumptively identify S. saprophyticus
-S. saprophyticus is resistant to Novobiocin while S. aureus and S.epidermidis are sensitive

4. Antimicrobial Susceptibility

Penicillin- for those that do not produce B-lactamase

Methicillin- for penicillin-resistant staphylococci
Vancomycin- antimicrobial of choice for endocarditis due to MRSA (methicillin resistan tS. aureus) and MRSE (methicillin-
resistant S. epidermidis)
- has adverse effects; use is reserved for systemic infections such as bacteremia, endocarditis, and pneumonia
*Back-up antibiotic:Linezolid (for most resistant)
 FAMILY MICROCOCCACEAE

S.aureus S.epidermidis S.saprophyticus

Hemolysis Beta Non-hemolytic Nonhemolytic

Coagulase + - -

Heat Stable Dnase + - -

Mannitol Fermentation + - Variable

Novobiocin S S R
 FAMILY MICROCOCCACEAE
CATALASE
(+) (-)
Staphylococcus Streptococcus

-
+ COAGULASE
Coagulase Negative Staphy (CONS)
S. aureus

Novobiocin

S R
S. epidermidis S. saprophyticus
 FAMILY STREPTOCOCCACEAE
General Characteristics

• G (+) cocci arrange in pairs or chains

• Facultative anaerobes
• Catalase (-)
• Except for Viridans group and S. pneumonia, all have C
carbohydrate in their cell wall
• Need blood or enriched media for isolation
 FAMILY STREPTOCOCCACEAE
Basis for Classification:
A. Hemolytic Patterns on BA
1. beta - complete iysis of rbc around colony; clear area around colony
2. alpha - partial lysis of rbe around colony: greenish discoloration of area
around colonyre
3. nonhemolytic / gamma -no lysis of rbc around colony; no change in agar
4. alpha-prime/ wide-zone - small area of intact rbc surrounded by a wider
zone of complete hemolysis
 FAMILY STREPTOCOCCACEAE
Basis for Classification:
B. Physiologic Characteristics
1. Pyogenic - produce pus; mostly beta-hemolytic; majority of Lancefield
groups
2. Lactic acid production-found in dairy products; nonhemolytic;
Lancefield group N
3. Enterococci - normal flora of the human intestine
4. Viridians - normal flora of the upper respiratory tract; not part of
Lancefield's classification; often seen as opportunistic pathogens; do
not have a C carbohydrate
 FAMILY STREPTOCOCCACEAE
Basis for Classification:

C. Lancefield Classification (Serologic groups according to type

of C carbohydrate present)
-C carbohydrate can be extracted from the streptococcal cell wall using acid
and heat
-soluble antigen is used to immunize rabbits to obtain antisera to the various
C carbohydrate groups

See table…..
 FAMILY STREPTOCOCCACEAE
Basis for Classification:
Common Terms Lancefield Group Species Hemolytic pattern
Group A Strep A Streptococcus pyogenes Beta
Group B Strep B Streptococcus agalactiae Beta
Group C Strep C Streptococcus equisimillis Beta
Streptococcus equi

Group D non-enterococcus D Streptococcus bovis Alpha or nonhemolytic

Streptococcus equines

Enterococcus D Enterococcus faecalis Alpha, Beta ,None

Enterococcus faecium
Enterococcus durans

Pneumococcus None Streptococcus pneumonia Alpha

Viridans Strep None Streptococcus anginosus Alpha, Alpha-prime, none
Streptococcus sanguis
Streptococcus mitis
Streptococcus mutans
 FAMILY STREPTOCOCCACEAE
Basis for Classification:
D. Biochemical Characteristics
1. Vancomycin
▪ effective for g+ organisms
▪ resistance to VA is now seen more commonly; g+ orgs should be routinely
tested for VA susceptibility
▪ significant number of Enterococcus demonstrate resistance to VA

2.Bacitracin Susceptibility
▪ Also known as Taxo A
▪ to differentiate S. pyogenes from other B-hemolytic groups;
▪ selectively inhibits the growth of group A strep
▪ positive reaction : zone of inhibition around the disk presumptive
ID
 FAMILY STREPTOCOCCACEAE
Basis for Classification:
D. Biochemical Characteristics
3. SXT Susceptibility (sulfamethoxazole and trimethoprim)
▪ to screen for S. pyogenes from throat cultures
▪ used in conjunction with Bacitracin to improve the accuracy of group A
identification
▪ positive reaction: zone of inhibition around the disk

4. PYR hydrolysis
▪ L-pyrrolidonyl-B-naphthylamide or L-pyroglutamic acid-B-naphthylamide
▪ for S. pyogenes : more specific than bacitracin susceptibility for
Enterococcus: as specific as bile esculin and NaCl broth
 FAMILY STREPTOCOCCACEAE
Basis for Classification:
D. Biochemical Characteristics

5. Hippurate hydrolysis
▪ to differentiate S. agalactiae from other B-hemolytic strep

6. CAMP (Christie, Atkins, Munch-Petersen)

▪ to differentiate S. agalactiae from other B-hemolytic strep;
presumptive ID
▪ S. agalactiae produces a CAMP factor that enhances the
lysis of rbc by staphylococcal B-lysin
 FAMILY STREPTOCOCCACEAE
Basis for Classification:
D. Biochemical Characteristics

6. CAMP (Christie, Atkins, Munch-Petersen)

Methods:
a. use of a B-lysin producing strain S.
aureus(arrowhead enhanced hemolysis)
b. use of a disk impregnated with the B-lysin
(enhanced hemolysis)
c. rapid CAMP test: use of extracted B-lysin from
S. aureus(enhanced hemolysis)
 FAMILY STREPTOCOCCACEAE
Basis for Classification:
D. Biochemical Characteristics
7. Optochin (ethylhydro cuprein hydrochloride)
▪ Also known as(Taxo P)
▪ to differentiate S pneumoniae from other alpha-hemolytic strep; presumptive ID
▪ positive reaction:zone of inhibition around the disk

8. Bile solubility
▪ S. pneumoniae has auto catalytic enzyme
▪ in the presence of bile salts such as Na deoxycholate, the cell wall lyses
▪ positive reaction: suspension becomes çlear
 FAMILY STREPTOCOCCACEAE
Basis for Classification:
D. Biochemical Characteristics
9. Bile esculin
▪ to differentiate group D strep and Enterococcus from other
g+ cocci
▪ group D strep and Enterococcus can grow in the presence of
40% bile and hydrolyze esculin
▪ positive reaction: darkening of medium ; (-) orange

10. Growth in 6.5% NaCl

▪ for identifying Enterococcus
▪ positive reaction: turbidity
 FAMILY STREPTOCOCCACEAE
Basis for Classification:
D. Biochemical Characteristics
S. pyogenes S. agalactiae Group D Strep Enterococcus S. pneumoniae Viridans
Hemolysis B B A,N A,B,N A A,A’,N
Bacitracin S R
SXT R R
PYR + - - + - -
Hippurate - +
CAMP - +
Optochin R R S R
Bile solubilty - - + -
Bile Esculin + + - -
6.5 % NaCl - + - -
 FAMILY STREPTOCOCCACEAE
Clinically Significant
1. Streptococcus Pyogenes / Group A Streptococci
A. Virulence Factors:

1. M protein
• resistance to phagocytosis and adherence to mucosal cells
• more than 80 different serotypes

2. Streptolysin O
• oxygen labile; active only in reduced form or anaerobic condition
• highly antigenic; ASTO indicates recent infection with S. pyogenes
• lyses leukocytes, platelets, rbc –
 FAMILY STREPTOCOCCACEAE
Clinically Significant
1. Streptococcus Pyogenes / Group A Streptococci
A. Virulence Factors:
3. Streptolysin S
• Oxygen stable; responsible for hemolysis when incubated aerobically
• Nonantigenic
4. DNases (A, B, C, D) -(streptodornase) antigenic
5. Streptokinase - lyses fibrin clots
6. Hyaluronidase - spreading factor
 FAMILY STREPTOCOCCACEAE
Clinically Significant
1. Streptococcus Pyogenes / Group A Streptococci
A. Virulence Factors:
7. Erythrogenic toxin
• 3 types(A, B, C); causes the spreading rash in scarlet fever
• also known as streptococcal pyrogenic exotoxin

8. Protein F
• fibronectin-binding protein; adhesion molecule
9. Hyaluronic acid capsule - prevents opsonization; hides antigens
1. Streptococcus Pyogenes / Group A Streptococci
Clinical Infections
1. pharyngitis and tonsillitis/ "strep throat"

▪ Usually in children 5-15 years old; incubation period 1-4

days; spread by droplets and close contact
▪ Manifests as sore throat, malaise, fever, headache, inflamed
tonsils and pharynx; swollen and tender cervical lymph
nodes
▪ Definitive diagnosis relies on a throat culture; 1/3 of those
with sore throat have a throat culture positive for S.
pyogenes
1. Streptococcus Pyogenes / Group A Streptococci
Clinical Infections
2. pyodermal infections

• impetigo - localized skin disease; small vesicles that

progress to weeping lesions; seen in children 2-5 yrs old
• Erysipelas- – infection of the skin and subcutaneous
tissues; erythematous lesion; seen in elderly patients
• cellulitis- develops following deeper invasion by strep;
life-threatening with bacteremia or sepsis present
• scarlet fever- red rash appears on the upper chest,
spreads to the trunk and extremities; asso w/
erythrogenic toxin
1. Streptococcus Pyogenes / Group A Streptococci
Clinical Infections
3. streptococcal toxic shock syndrome
• same toxin associated with scarlet fever

4. necrotizing fasciitis
• "flesh-eating disease;" Type 2 NF )
1. Streptococcus Pyogenes / Group A Streptococci
Complications
1. rheumatic fever
• complication of S. pyogenes pharyngitis
• fever, inflam'n of the heart, joints, blood vessels; most
serious result is a progressive damage to the heart valves
• attacks begin within a month after infection; repeated
infections may produce further valve damage
pathogenesis:
✓antigenic cross-reactivity between streptococcal antigens and heart
tissue
✓direct toxicity due to bacterial exotoxins
✓actual invasion of the heart tissues by the organism
1. Streptococcus Pyogenes / Group A Streptococci
Complications
2. acute glomerulonephritis (AGN)
• may occur after a cutaneous or pharyngeal infection;
more common in children than adults
• pathogenesis: antigen-antibody complexes deposit in the
glomerulus, complement is fixed, inflammatory response
causes damage to the glomerulus and impairment o
kidney function
1. Streptococcus Pyogenes / Group A Streptococci
Treatment

penicillin (drug of choice)

erythromycin (for patients allergic to penicillin)
1. Streptococcus Pyogenes / Group A Streptococci
Lab Diagnosis
• mall, transparent, smooth colonies on BA
• BA with SXT is used for better recovery from throat
cultures
✓Gram-positive cocci ;Beta hemolytic ;Catalase (-)
Definitive ID:
serologic typing (Lancefield typing)
Bacitracin (S) CAMP (-)
Hippurate hydrolysis (-)
PYR(+)
*Immunologic tests to detect past infection with S. pyogenes: Antistreptolysin-0
(ASO); Anti-DNase ;Antistreptokinase ;Antihyaluronidase
2. Streptococcus agalactiae/Group B Streptococci

3 serotypes based on capsular polysaccharides ( I, II, II)

A. Virulence Factors: capsule with sialic acid
(prevents phagocytosis) CAMP factor
hyaluronidase, hemolysin, DNase,
neuraminidase, protease
2. Streptococcus agalactiae/Group B Streptococci

B. Clinical Infections:
Neonates
1. early onset infection - w/in 3 days after
birth - usually manifests as pneumonia or
meningitis with bacteremia
2. late onset infection - between 1 week and
3 months after birth – usually ımeningitis
2. Streptococcus agalactiae/Group B Streptococci

B. Clinical Infections:
Adults
1. women after childbirth-abortion- endometritis/ wound
infection
2. elderly with serious underlying disease or
immunodeficiency

C. Treatment: penicillin; ampicillin + aminoglycoside

2. Streptococcus agalactiae/Group B Streptococci

• grayish white mucoid colonies surrounded by a small zone of B-

hemolysis in BA

Gram-positive cocci
Beta hemolytic
Catalase (-)
Definitive ID: serologic typing (Lancefield typing)
CAMP (+) :increased lysis in the shape of arrowhead
Bacitracin (R)
Hippurate hydrolysis (+)
PYR (-)
3. Group D Streptococcus

previously subdivided into two groups:

*enterococcal group - now placed in a new genus Enterococcus
*nonenterococcal group - remained part of the group D strep

Species:
S. equinus
S. bovis -nonenterococcal isolate but is found in the intestinal tract
- bacteremia has been associated with gastrointestinal tumors
Clinical Infections: bacterial endocarditis, UTI, abscesses, wound
infections
3. Group D Streptococcus
Treatment/Laboratory Diagnosis
B. Treatment: Group D strep is susceptible to
penicillin (Enteroccus is resistant)

ID:
Gram positive cocci
serotyping for S. bovis
Alpha or nonhemolytic
Bile esculin (+)
PYR(-) Catalase (-)
6.5% NaCl (-)
4. Enterococcus
• previously known as group D Streptococcus enterococcus
group
• found in intestinal tract
• associated with UTI in hospitalized patients, endocarditis,
bacteremia, wound infections

A. Clinical Infections: same with group D (nonenterococci)

4. Enterococcus
Treatment/ Laboratory Diagnosis
resistant to several of the commonly used antibiotics

ID:
Gram positive cocci
Bile esculin (+)
Alpha, B, nonhemolytic
6.5 % NACI (+)
Catalase (-)
PYR (+)
5. Streptococcus pneumoniae
✓may be found as normal flora of URT

Antigenic structure:
1. C substance in the cell wall (similar to C carbohydrate of
Lancefield groups)
2. Capsular antigens (82 different capsular types)
- capsule is antigenic and identified with appropriate antiserum
-Neufeld Quellung rxn: capsule swells in the presence of specific
anticapsular serum

A. Virulence Factors: Capsular Polysaccharide

Hemolysin; Ig A protease; Neuraminidase & Hyauronidase
5. Streptococcus pneumoniae
Clinical Infections
1. pneumonia

- lobar pneumonia (rusty-brown sputum

- caused by types 1, 2, 3
- - predisposing factors: alcoholism, malnutrition, anesthesia, viral
infections of the respiratory tract

2. otitis media

- S. pneumoniae is the most common cause otitis media in

children < 3yrs old
5. Streptococcus pneumoniae
Clinical Infections
3. meningitis - most common cause of bacterial meningitis in
adults

4. endocarditis, peritonitis, bacteremia

C. Treatment: penicillin; erythromycin; chloramphenicol

5. Streptococcus pneumoniae
Laboratory Diagnosis
Gram positivediplococci(lancet-shaped)
Catalase (+)
Quellung Capsular Swelling (+)
Optochin (S)
Bile solubility (+)

*capsule can also be demonstrated by a capsule

stain
6. Viridans Streptococci
Clinical Infections
✓lack Lancefield group antigens and do not meet the criteria for S.
pneumoniae
✓ID of the viridans strep to the species level is difficult
✓oropharyngeal commensals but are opportunistic pathogens

1.-subacute bacterial endocarditis - most common cause of SBE

dextran allows adherence to vascular endothelium

2. meningitis, dental caries, abscesses, osteomyelitis, empyema B.

✓Treatment: penicillin
6. Viridans Streptococci
Lab Diagnosis

Gram positive cocci

Bile esculin (-)
Bile solubility (-)
alpha or nonhemolytic
Optochin (R)
PYR (+) Catalase (-)
6.5 % NaCl (-)
 Genus Abiotrophia

✓Formerly known as Nutritionally-variant Streptococei /

Pyridoxal-dependent/ vitamin B6-dependent / Thiol-
dependent/ Symbiotic Streptococci
✓they satellite around colonies of organism that produces
pyridoxal: staphylococci, E. coli, Klebsiella spp, Enterobacter,
and yeasts
✓Gram positive cocci (may be Gram- variable and pleomorphic)
✓ part of the normal oral flora but can cause endocarditis, otitis
media, wound infections
 Genus Abiotrophia
Lab Diagnosis

• use of pyridoxal (Vit B6) supplemented medium or plated with a

staphylococcal streak (cross-streaking)

PYR (+)
Bile esculin (-)
6.5 % NaCI(-)