Transcript Pathology Resident Microbiology Lecture Series

MLAB 2434: MICROBIOLOGY
KERI BROPHY-MARTINEZ
Staphylococci
TAXONOMY
Family:
Micrococcaceae
Genus:
 Staphylococcus
Coagulase positive
Coagulase negative

 Micrococcus
GENUS STAPHYLOCOCCUS
Coagulase positive

S. aureus
Coagulase negative
S. epidermidis
 S. saprophyticus
 S. haemolyticus

STAPHYLOCOCCUS:
GRAM REACTION AND MORPHOLOGY
Gram-positive spherical cells
(0.5-1.5 mm) in singles, pairs,
and clusters
 Appear as “bunches of
grapes”

Gram-stained smear of
staphylococci from colony
Scanning electron micrograph of
staphylococci
STAPHYLOCOCCUS:
GENERAL CHARACTERISTICS

Nonmotile

Non–spore-forming

Nonencapsulated

Catalase-producing

Oxidase: negative

Glucose fermenters

Primarily aerobic, some facultatively anaerobic
STAPHYLOCOCCUS:
GENERAL CHARACTERISTICS (CON’T)
Bacitracin resistant
 Grow on agar that contains peptone
 Inhibited by media that has high bile salt concentration
 Some are ß-hemolytic
 Colony morphology: buttery looking, cream or white
colored

STAPHYLOCOCCUS AUREUS
Primary pathogen of the genus
 Habitat:

Anterior nares (carriers)
 Colonization: axilla, vagina, pharynx


Produce superficial to systemic infections
Skin
 Bacterial sepsis
 Hospital acquired infections

STAPHYLOCOCCUS AUREUS

Mode of transmission
Traumatic introduction
 Direct contact with infected person
 Inanimate objects


Predisposing conditions
Chronic infections
 Indwelling devices
 Skin injuries
 Immune response defects

STAPHYLOCOCCUS AUREUS

Infection will elaborate inflammatory response with
GPC accumulating as pus

Pus: mix of active and inactive neutrophils, bacterial cells
and extravascular fluid
VIRULENCE FACTORS OF S. AUREUS




Enterotoxins
Cytolytic toxins
Enzymes
Protein A
VIRULENCE FACTORS: ENTEROTOXINS

Enterotoxins:

Heat-stable exotoxins that cause diarrhea and vomiting
Exotoxin: protein produced by a bacteria and released into
environment
o
 Heat stable @ 100 C for 30 minutes


Implications
Food poisoning
 Toxic shock syndrome
 Pseudomembranous enterocolitis

TYPES OF ENTEROTOXINS

Exfoliatin


Epidermolytic toxin
TSST-1: Toxic shock syndrome toxin-1
Multisystem disease
 Stimulates T cell production & cytokines


Cytolytic Toxins
Affects RBCs and WBCs
 Hemolytic toxins: alpha, beta, gamma, delta
 Panton-Valentine leukocin, lethal to WBCs

VIRULENCE FACTORS: EXTRACELLULAR ENZYMES

Hyaluronidase:


Staphylokinase:


Fibrinolysin which allows spread of infection
Coagulase:


Hydrolyzes hyaluronic acid in connective tissue
allowing spread of infection
Virulence marker
Lipase:

Allows colonization by acting on lipids present on the
surface of the skin.
VIRULENCE FACTORS: EXTRACELLULAR
ENZYMES (CON’T)

Penicillinase:


DNase:


Confers resistance
Degrades DNA
Beta-lactamase:

Cuts the beta lactam wall of certain antibiotics
VIRULENCE FACTORS: PROTEIN A
 Protein
A:
 Found in cell wall
 Binds to Fc part of IgG
 Blocks phagocytosis
STAPHYLOCOCCUS AUREUS: CLINICAL
INFECTIONS

Skin and wound

Impetigo

Furuncles/Boils (Infection
of hair follicles usually in
areas that sweat)

Carbuncles (clusters of
boils)

Surgical wound infections
Bullous impetigo
STAPHYLOCOCCUS AUREUS:
CLINICAL INFECTIONS (CON’T)

Skin and wound


Scalded skin syndrome= Ritter’s disease

Extensive exfoliative dermatitis

Young children and newborns
Toxic Shock Syndrome
 Multisystem disease
 Caused by TSST-1
 Affects women, men, and children
STAPHYLOCOCCUS AUREUS: CLINICAL
INFECTIONS
 Food
poisoning
 Source is infected food handler
 Enterotoxin A the most common cause
 Foods affected include meat, dairy products,
bakery goods with cream fillings, and salads
made with eggs and mayonnaise.
COAGULASE-NEGATIVE STAPHYLOCOCCI
 Found
as indigenous flora
 Presence can indicate contamination
 Seeing an increase due to prosthetic devices,
catheters and immunocompromised
 Abbreviated CNS or CoNS
COAGULASE-NEGATIVE STAPHYLOCOCCI
 Habitat:

Skin and mucous membranes
 Common
human isolates
 S. epidermidis
 S. saprophyticus
 S. haemolyticus
COAGULASE-NEGATIVE STAPHYLOCOCCI:
STAPHYLOCOCCUS EPIDERMIDIS
 Predominantly
hospital acquired infections
 Skin flora gets introduced by catheters, heart
valves, CSF shunts
 Produces a slime layer that helps adherence to
prosthetics and avoidance of phagocytosis
 UTIs are a common result
COAGULASE-NEGATIVE STAPHYLOCOCCI:
STAPHYLOCOCCUS SAPROPHYTICUS

UTIs in young sexually active women

Due in part to increased adherence to epithelial cells lining
the urogenital tract
Rarely present in other skin areas or mucous
membranes
 Urine cultures


If present in low amounts, it is still considered significant
COAGULASE-NEGATIVE STAPHYLOCOCCI:
STAPHYLOCOCCUS HAEMOLYTICUS

Habitat: skin and mucous membranes

Rarely implicated in infections

Associated with wound infections, bacteremia, and
endocarditis
BREAK TIME!!!
LABORATORY DIAGNOSIS: SPECIMEN COLLECTION
AND HANDLING



Samples must be taken from the actual site of infection
Prevent delay in transport of collected material from
infected sites
Transport in appropriate collection device that would
prevent drying and minimize growth of contaminating
organisms
LABORATORY DIAGNOSIS:
DIRECT SMEAR EXAMINATION
Microscopic Examination
o
Gram reaction
o
o
Cell arrangement
o
o
Gram-positive cocci
Pairs and clusters
Presence/Absence of PMNs
o
Numerous polymorphonuclear cells
(PMNs)
Insert Figure 10-1
LABORATORY DIAGNOSIS:
CULTURAL CHARACTERISTICS Staphylococcus
aureus
 Colony morphology
Smooth, butyrous,
white to yellow,
creamy
Grow well @ 18-24
hours
S. aureus may
produce hemolysis
on blood agar
S. aureus
LABORATORY DIAGNOSIS: CULTURAL
CHARACTERISTICS
 S.
epidermidis

Smooth, creamy, white

Small-to mediumsized, usually nonhemolytic
 S.

saprophyticus
Smooth, creamy, may
produce a yellow
pigment
IDENTIFICATION TESTS: CATALASE


Principle: tests for enzyme catalase
2 H 2O 2
2 H2O + O2
Procedure
 Smear a colony of the organism to a slide
 Drop H2O2 onto smear
 Observe
CATALASE TEST: INTERPRETATION

Presence of bubbles
Positive
Staphylococci

Absence of bubbles
Negative
Streptococci
IDENTIFICATION TEST: SLIDE COAGULASE TEST



Differentiates members within the Staphylococci
Detects clumping factor found in S. aureus
Procedure
 Place a drop of sterile water on a slide and emulsify a colony
 Add a drop of rabbit plasma to the suspension
 Observe
 Agglutination = Positive
 No agglutination= Negative
IDENTIFICATION TESTS: COAGULASE TEST
•Detects the extracellular enzyme “free
coagulase” or staphylocoagulase
•Causes a clot to form when bacterial cells
are incubated with plasma
•Procedure
•Inoculate rabbit plasma with
organism and incubate at 35-37 0 C
•Observe at 30 minutes for the
presence of a clot
•Continue for up to 24 hours, if
needed
IDENTIFICATION TESTS: RAPID COAGULASE
TEST

Latex Agglutination Assays

Detects cell-bound “clumping factor,”
protein A or a combination of both

Procedure
 Varies depending on kit type
 Positive reaction demonstrated by
agglutination
NOVOBIOCIN SUSCEPTIBILITY TEST

Test to differentiate coagulasenegative staphylococci from
S.saprophyticus from urine
samples
S. saprophyticus is resistant (top)
 Other CNS are susceptible

MICROCOCCUS
Rarely produces disease
 Found in environment and indigenous skin flora
 Catalase +
 Coagulase =
 Produces yellow pigment
 Microdase disc differentiate between Staph &
Micrococcus

Schematic Diagram for Identifying
Staphylococcal Species
ANTIMICROBIAL SUSCEPTIBILITY

For non–beta-lactamase producing S. aureus

Use pencillin


Penicillinase-resistant synthetic penicillins (methicillin, nafcillin,
oxacillin, dicloxacillin)
Beta-lactamase producers break down the beta-lactam
ring of penicillin so it inactivates antibiotic before it acts
on bacterial cells
METHICILLIN-RESISTANT STAPHYLOCOCCI
MRSA
 Methicillin-resistant S. epidermidis



MRSE
Infection control
Barrier protection
 Contact isolation
 Handwashing


Treat with vancomycin

Test for susceptibility with cefoxitin disk
METHICILLIN-RESISTANT STAPHYLOCOCCI
(CONT’D)
 mecA
gene
 Encodes penicillin-binding proteins (PBPs)
 Causes drug ineffectiveness

Gold standard
Nucleic acid probe or PCR for the mec A
gene
VANCOMYCIN-RESISTANT STAPHYLOCOCCI
VRSA= vancomycin resistant Staphylococcus aureus
 VISA= vancomycin intermediate Saphylococcus aureus


Detection

Vancomycin screening media
ANTIMICROBIAL SUSCEPTIBILITY

Macrolide Resistance
Clindamycin sensitivity often requested by physician to treat
Staph skin infection. Referred to as “D” test
 Clindamycin resistance is often inducible meaning it only is
detectable when bacteria are also exposed to erythromycin

SUMMARY MICROCOCCACEAE
Staph. aureus
Colony
Morphology
Opaque,
smooth, raised,
entire, whitegolden(cream)
Hemolysis
Most are beta
hemolytic
GPC in
clusters, pairs,
short chains or
singly
Pos
Fermenter
Non-hemolytic
Staph.
saprophyticus
Opaque,
smooth, raised,
entire,
butyrous,
glossy, whiteyellow
Non-hemolytic
GPC in
clusters, pairs,
short chains or
singly
Pos
Fermenter
GPC in
clusters, pairs,
short chains or
singly
Pos
Fermenter
GPC in pairs
and tetrads
Neg
Neg
Neg
Pos
Resistant
Resistant
Resistant
Sensitive
Pos
Neg
Neg
N/A
Pos
Neg
Neg
Neg
Gram
morphology
Catalase
Glucose
fermentation
Modified
Oxidase
Bacitracin
susceptibility
(Taxo A
0.04U)
Coagulase
Production
(tube)
Clumping
factor (slide or
latex
Coagulase test)
Staph.
Epidermidis
Opaque,
smooth, raised,
entire, graywhite
Micrococcus
Opaque,
smooth, raised,
white, bright
yellow
Non-hemolytic
Pos
Oxidizer
REFERENCES

Engelkirk, P., & Duben-Engelkirk, J. (2008). Laboratory Diagnosis of Infectious Diseases:
Essentials of Diagnostic Microbiology . Baltimore, MD: Lippincott Williams and Wilkins.

http://archive.microbelibrary.org/ASMOnly/Details.asp?ID=2037

http://brawlinthefamily.keenspot.com/gallery/2009-10-18-breaktime/

http://ericaandkevin.pbworks.com/w/page/5827086/Gram-Stain-and-Other-Tests



http://faculty.matcmadison.edu/mljensen/111CourseDocs/111Review/Unit2Reviews/micrococcace
ae_answers.htm
http://jeeves.mmg.uci.edu/immunology/Assays/LatexAgglut.htm
Mahon, C. R., Lehman, D. C., & Manuselis, G. (2011). Textbook of Diagnostic Microbiology (4th
ed.). Maryland Heights, MO: Saunders.