CATALASE-POSITIVE,

GRAM-POSITIVE COCCI

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¢ Medically important Genera: Staphylococcus

¢ General characteristics
A. Staphylococcus — Gram-positive spherical cells (0.5-1.5 mm) in singles, pairs, and
clusters
B. Micrococcus — Appear as “bunches of grapes”

C. Planococcus

D. Stomatococcus

Scanning electron micrograph of Gram-stained smear of

staphylococci staphylococci from colony

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 Staphylococcus
¢ General characteristics
— Catalase producing
— Facultative anaerobe except S. sacharrolyticus
— Glucose fermenter (anaerobically)
— Non-motile, non spore-forming, nonencapsulated
— Halophilic (7.5-10% NaCl)
— Differentiated by coagulase test
— Reduces nitrates to nitrites
— Modified oxidase (-)
— Lysostaphin (Susceptible)
— Bacitracin (Resistant)

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Staphylococcus
COAGULASE POSITIVE STAPHYLOCOCCI
¢ General characteristics
— Species of Staphylococci are initially differentiated by COAGULASE TEST ¢ S. aureus
¢ S. intermedius Human
— SLIDE TEST/Screening test pathogens
¢ detects clumping factor/bound coagulase
¢ S. lugdunensis
— TUBE TEST/Confirmatory test
¢ detects free coagulase
¢ reacts w/ CRF or coagulase reacting factor that resembles thrombin & converts ¢ S. hyicus
fibrinogen to fibrin Animal-associated
¢ S. delphini species
— For the vast majority of clinical laboratory situations, coagulase-positive
isolates from human sources are considered to be S. aureus, because other ¢ S. schleiferi
species are often animal-associated.

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 Staphylococcus Micrococcus
¢ General characteristics ¢ Key characteristics
— Staphylococci that do not produce coagulase are referred to as Coagulase-negative
staphylococci (CoNS)
— Gram positive cocci
— Most clinically and commonly recovered significant CoNS species are:
— Found as resident flora of the skin, mucosa & oropharynx
¢ S. epidermidis — Strict Aerobes
¢ S. saprophyticus
— Nonmotile, non-sporulated, non-capsulated
¢ S. haemolyticus

¢ S. lugdunensis
— Catalase producing
— More than 40 CoNS exist and several species have been isolated from humans, — Coagulase negative
usually from the skin and mucous membranes. — Modified oxidase (+)
— Some species are found on very specific sites such as the head (S. capitis) and ear — Lysostaphin (Resistant)
(S. auricularis) — Bacitracin (Susceptible)

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DIFFERENTIATION OF STAPHYLOCOCCUS FROM MICROCOCCUS

Test Staphylococcus Micrococcus

Catalase (+) (+)
Aerobic growth (+) (+)
Anaerobic growth (+) (-)
Glucose Utilization Fermentative Oxidative
Modified Oxidase (-) (+)
Lysostaphin Susceptible Resistant
(200ug/ml)
Bacitracin (0.04 U) Resistant Susceptible

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¢ Clinical Significant Staphylococcus species

A. Staphylococcus aureus
B. Staphylococcus epidermidis
C. Staphylococcus saprophyticus
D. Staphylococcus haemolyticus
E. Staphylocccus lugdunensis
F. Methicillin-resistant Staphylococcus aureus
G. Rarely isolated CoNS

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 ¢ VIRULENCE FACTORS/ Pathogenic Determinant
A. Staphylococcus aureus
— Most clinically significant specie of Staphylococci
— Appears as medium to large colonies, 2 to 3mm
diameter with a convex, creamy appearance.
— Edge is entire and colonies may be pigmented
white to golden yellow
— Most strains exhibit a narrow zone of beta hemolytic while some
are non-hemolytic
— Important cause of nosocomial infection
— Responsible for a number of infections both relatively mild and life
threatening

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¢ Virulence Factors:
— Protein A
¢ Found in cell wall

¢ Binds to Fc part of IgG

¢ Blocks phagocytosis

— Enterotoxin (A-E & G-J)

¢ Heat-stable exotoxins that cause diarrhea and vomiting
¢ Exotoxin: protein produced by a bacteria and released into environment

¢ Heat stable @ 100o C for 30 minutes

¢ Implications

¢ Food poisoning

¢ Toxic shock syndrome

¢ Pseudomembranous enterocolitis

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 ¢ Virulence Factors: ¢ Virulence Factors:
— Enterotoxin A & D — Exfoliatins
¢ Associated with staphylococcal-related food poisoning ¢ Also known as Epidermolytic toxin (A & B)
¢ Hydrolyze tissue through cleavage of stratum granulosum
— Enterotoxin B
¢ Associated with SSS/Ritter Lyell Disease
¢ Associated with staphylococcal enterocolitis
— Enterotoxin F — Cytolytic toxins
¢ Also known as TSST-1 ¢ Affects RBCs and WBCs
¢ Associated with Toxic Shock Syndrome (TSS)
¢ Hemolytic toxins: alpha, beta, gamma, delta
¢ Panton-Valentine leukocidin: lethal to PMNs

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¢ Virulence Factors: ¢ Virulence Factors:

— Enzymes — Enzymes
¢ Fibrinolysin ¢ Lipase
¢ Hydrolyze lipids in plasma & skin.
¢ Also known as Staphylokinase
¢ Enable staphylococci to colonize certain body areas
¢ Dissolves fibrin clot & may enable infection
¢ Associated with initiation of skin infections such as boils, carbuncles &
to spread once the clot is dissolve furuncles
¢ Coagulase ¢ Hyaluronidase

¢ Virulence marker ¢ Hydrolyzes hyaluronic acid in connective tissue

¢ Conversion of fibrinogen to fibrin allowing spread of infection

¢ Deoxyribonuclease (Dnase)
¢ May coat neutrophils with fibrin formed
¢ Degrades DNA
to protect organism from phagocytosis

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Infection caused by Staphylococcus aureus 1.Boils (pigsa)
— Due to lipase which promotes hydrolyzes of lipid component of the ski
2.Pimples
— Foliculitis – infection or inflammation of hair follicle
¢ Furuncle

¢ Carbuncle

3.Stye (kuliti)
— Infection of upper or lower eyelids
4.Cellulitis
— Infection of the deeper tissue
5.Meningitis
— Presence of Protein A
6.UTI

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 7.SSS/Ritter Lyell Disease (RLD)
— Due to exfoliatin/epidermolytic toxin
— Extensive exfoliative dermatitis that occurs primarily in
newborn
— Cases of SSS in adults occur most commonly in patients with chronic
renal failure and immunocompromised patients
— Severity of disease varies from being a localized skin lesion in the form
of bullous impetigo to a more extensive generalized condition,
characterized by cutaneous erythema followed by a profuse peeling of
the epidermis
— Typical pattern in which erythema occurs is origination from the face,
neck, axillae, and groin and extension to the trunk and extremities.
— Duration of disease is brief, usually 2-4 days.
— Toxin is metabolized and excreted by the kidneys

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8. Toxic Shock Syndrome (TSS) 9. Food Poisoning

— A clinical syndrome characterized by hypotension, fever, desquamation of the palms
and soles, fever, chills, headache, and vomiting
— First characterized by Todd in 1979
— Associated with highly absorbent tampon use (in 1979 & 1980s,
91% of TSS cases were menstruating-associated)
— Staphylococcal TSS generally results from a localized infection by S. aureus; only the
toxin TSST-1 is systemic
— Cultures of focal lesions may yield S. aureus, but blood cultures are usually negative.
— Supportive therapy to replace vascular volume loss is given, along with appropriate
antimicrobial therapy
— Preventive measures, such as use of minimum absorbency tampons and warning label
requirement by U.S. FDA for tampons have greatly reduced the risk of TSS.
— S. aureus enterotoxins associated with food poisoning:
¢ Enterotoxin A (78%)
¢ Enterotoxin D (38%)
¢ Enterotoxin B (10%)
— Source of contamination is usually an infected food handler
— Disease occurs when food becomes contaminated with the enterotoxins by improper handling and then
improperly stored, which allows growth of bacteria and resulting toxin production
— Foods commonly contaminated include salads with mayonnaise and eggs, meat products, poultry, egg
products, bakery products, sandwich fillings and dairy products
— Symptoms appear rapidly approx. 2 to 8 hours after ingestion of food
— Although fever is usually absent, there is nausea, vomiting, abdominal pain, severe cramping and diarrhea
are common.
— Death from staphylococcal food poisoning is rare, although such cases have
occurred among elderly patients, infants and debilitated patients.

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 10. Osteomyelitis Staphylococcus epidermidis
— Infection in the bone marrow
— Seen in children as a complication in patients with DM or Artherosclerosis, and as a result
of trauma or surgery ¢ Formerly known as Staphylococcus albus
— S. aureus is the number 1 cause of osteomyelitis in US ¢ Normal flora of the skin & mucous membranes
¢ Coagulase (-)
11. Impetigo
— Rashes all over the body
¢ Novobiocin (S)
¢ Dnase (-)
12. Staphylococcal pneumonia ¢ Most commonly encountered & less virulent than S. aureus
— has been known to occur secondary to influenza virus infection ¢ Associated with the use of implants such as indwelling catheters
— develops as a contiguous, lower resp. tract infection or a complication of bacteremia, characterized by
multiple abscesses in the pulmonary parenchyma ¢ Opportunistic pathogen
— Infants and immunocompromised patients are most affected ¢ Produces a slime layer that helps adherence to prosthetics and avoidance of phagocytosis

13. Staphylococcal bacteremia ¢ Infections:

— observed among IV drug users — Bacteremia
— organism gain entry into the bloodstream via contaminated needles or from focal lesions
— Prostatic Valve Endocarditis
— Common cause of hospital acquired UTI

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Staphylococcus saprophyticus Staphylococcus haemolyticus

¢ Normal flora of the mucous membranes of the GUT ¢ Commonly isolated CoNS
¢ Habitat: skin and mucous membranes
¢ Associated with UTI in young sexually active women ¢ Rarely implicated in infections
¢ Associated with wound infections, bacteremia, and endocarditis
¢ Considered significant even if it is found in small number ¢ Some isolates are Vancomycin (R)
(less than 10,000 CFU/ml)
¢ Can be differentiated with S. epidermidis by Novobiocin test Staphylococcus lugdenensis
¢ Coagulase (-)
¢ Normal flora of the skin and GUT
¢ Novobiocin (R)
¢ Opportunistic pathogens
¢ Dnase (-) ¢ Coagulase (-)
¢ Novobiocin (S)
¢ Infections:
— Nosocomial bacteremia
— Endocarditis

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 Methicillin Resistant Staphylococcus aureus (MRSA) Rarely isolated CoNS
¢ S. auricularis
Normal flora of human & rare pathogen
¢ Oxacillin and cefoxitin are used to detect MRSA
—

¢ S. capitis
¢ Resistance is due to gene MecA which codes for altered — Normal flora of human scalp & usually nonpathogenic
S. conhnii
penicillin binding protein (PBP) ¢
— Normal flora of human skin & rare pathogen
¢ The altered PBP does not bind oxacillin thereby rendering it ¢ S. hominis
Normal flora of human skin & rare pathogen
ineffective ¢
—
S. schleiferi
¢ VANCOMYCIN – drug of choice for serious staphylococcal — Rare human pathogen in wound infection and bacteremia
¢ S. simulans
infection — Normal flora of human mucous memranes & rare pathogen
¢ S. warneri
— Normal flora of humans & usually nonpathogenic
¢ S. xylosus
— Normal flor of humans & rare cause of UTI

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Micrococcus
¢ Strict Aerobe
Oxidize Glucose
¢

¢ Catalase producers LABORATORY DIAGNOSIS OF

¢

¢
Modified (+)
Lysostaphin (Resistant)
STAPHYLOCOCCAL INFECTIONS
¢ Bacitracin (Susceptible)

Growth @ Blood Agar Plate

M. varlans M. luteus M. roseus

(yellow) (white) (pink)

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 ¢ 1. Gram Staining ¢ 2. Cultivation in Nutrient Agar
— Gram positive in singly, in pairs & clusters — S. aureus – golden yellow colonies
— S. albus – porcelain white colonies

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¢ 3. Catalase test ¢ 4. Coagulase test - pathogenicity test for S. aureus

— (+) – Staphylococcus & Micrococcus
— (-) – Streptococcus
— Rgt: 3% H2O2 à (+) bubbles/effervesence
— SCREENING/Slide
¢ Detect cell bound/clumping factor coagulase
¢ Rgt: Rabbit’s plasma or EDTA tube
¢ (+) clumping/clot formation

¢ (-) smooth suspension (no clot)

¢ Note: All negative coagulase slide test must be confirmed by the tube test

— CONFIRMATORY/Tube
¢ Detect free coagulase
¢ Rgt: 0.5ml of rabbit plasma & 0.5ml of Bacterial suspension/inoculum
¢ (+) solid coagulum

¢ (-) no clot formation

¢ Note: All tubes must be check for every 30 mins (1hour). If negative incubate RT
for 16-18 hrs

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 ¢ 5. Cultivation in BAP & CAP
— Small size/pin head colonies
— Margin is circular without interruption (entirely smooth)
— Dome shaped elevation
— Beta-hemolytic (some are gamma-hemolytic)
— Consistency/texture – butterlike/ glittering/
butyrous
— Density: opaque
— Odor: unwashed stocking odor

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 ¢ 6. Cultivation in CNA & PEA ¢ 7. Cultivation in MSA
— CNA
¢ Colistin-Nalidixic Agar
— Selective & differential medium
¢ BAP with two antibiotics — inhibitor of 7.5-10% NaCl
¢ Colistin (Polymyxin E) & Nalidixic

¢ Disruption of cell membrane of g (-) bacteria

— Indicator: Phenol Red
¢ Block DNA replication & membrane integrity in many gram negative bacteria — Incorporated CHO: Mannitol
¢ Fermenter = yellow
— PEA ¢ Non-fermenter = pink
¢ Phenyl Ethyl Alcohol Medium
¢ NA with 2 alcohols
¢ Phenyl & Ethylene

¢ Inhibit gram negative bacteria

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¢ 8. Dnase test ¢ 9. Voges Proskauer (VP) test

— DNase test agar is used to detect DNase activity in species of — Bacteria metabolized glucose by Butylene Glycol Pathway
aerobic bacteria — S. aureus, S. lugdunensis, S. haemolyticus, S. schleiferi (positive)
— Principle: Dnase medium using methyl green becomes colorless in
— S. intermedius (negative)
the presence of DNase
— (+) clear zone around the colonies
— (-) no clearing is observed
— Indicator: Methyl Green
— S. aureus(positive)
— S. intermedius & S. hyicus(negative)

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 ¢ 10. Novobiocin susceptibility test (5ug disk) ¢ 11. Gelatin liquefaction test
— S. saprophyticus (resistant) — Ability of the organism to produce gelatinase

— S. epidermidis, and majority of CoNS (susceptible) — Gelatin medium (butt/stab) – incubate for 12-24 hrs
— To confirm – refrigerate 1-6℃ liquefy (+)

— S. aureus (positive)
— S. intermedius & S. hyicus(negative)

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¢ 12. Modified Oxidase test (MOD) Coagulase

Nitrate Gelatin
Dnase
VP Reduction liquefaction
test test
— Test to differentiate Staphylococcus from Micrococcus test test

— Rgt: Tetramethylparahenylene diamine diHCl with S. aureus (+) (+) (+) (+) (+)
Dimethylsufoxide /Kovac’s reagent S. hyicus (+) (-) (-) (-) (-)
— (+) dark purple S. Intermedius (+) (-) (-) (-) (-)
— Micrococcus (positive)
— Staphylococcus (negative)
Novobiocin
test
S. aureus S
S. hyicus R
S. Intermedius S

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 Staphylococci Planococci Stomatococci Micrococci

Strict aerobe – + – +

Facultative + – + –
anaerobe
Motility – + – –

Growth on 6.5% + + – +
NaCl
Catalase + + v +

Anaerobic acid + – + –
from glucose

Lysostaphin S R R R

Bacitracin R v S S

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 References:
• 1. Pommerville, J. C. (2018). Fundamentals of microbiology. Jones &
Bartlett Learning.
• 2. Jorgensen, J. H., & Pfaller, M. A. (2015). Manual of clinical microbiology.
ASM Press.
• 3. Carroll, K. C., Landry, M. L., McAdam, A. J., Patel, R., Pfaller, M. A., &
Richter, S. S. (2019). Manual of clinical microbiology. ASM Press.
• 4. Fader, R. C., Engelkirk, P. G., & Duben-Engelkirk, J. L. (2019). Burton's
microbiology for the health sciences. Wolters Kluwer.
• 5. Delost, M. D. (2022). Introduction to diagnostic microbiology for the
laboratory sciences. Jones & Bartlett Learning.
• 6. Tille, P. M. (2022). Bailey & scott's diagnostic microbiology. Elsevier.
• 7. Riedel, S., Morse, S. A., Mietzner, T. A., & Miller, S. (2019). Jawetz,
Melnick & Adelberg's medical microbiology. McGraw-Hill Education.
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References:
• 1. https://asm.org/
• 2. https://microbiologyonline.org/index.php
• 3. https://learn.chm.msu.edu/vibl/
• 4. https://www.cartercenter.org/resources/pdfs/health/ephti/library/
lecture_notes/env_occupational_health_students/medicalbacteriology.pdf
• 5. https://www.atsu.edu/faculty/chamberlain/Website/links.htm
• 6. https://www.bioedonline.org/lessons-and-more/resource-
collections/micromatters-microbiology/
• 7. https://openstax.org/details/books/microbiology
• 8. https://www.edx.org/learn/microbiology
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