Staphylococci

Mahon Chapter 14
We encounter
staphylococci very
frequently
• As commensals

• As pathogens

• They are similar to Micrococcus

and recovered from similar sites
• Staph are GPC that divide in multiple planes, are
catalase positive, ferment sugars, are aerobic or
facultative anaerobes, nonmotile, and nonspore
forming
Why do these gram stains
look different?
Focus on the following
• Staphylococcus aureus
• S. epidermidis
• S. saprophyticus
• S. lugdunensis
• S. intermedius
• Micrococcus
S. intermedius *
S. lugdunensis *
Staphylococcus aureus
(halo)
• You may be colonized with S. aureus
• You may have had a staph infection
• These produce boils, folliculitis, furuncles, carbuncles and
purulent abscesses
• S. aureus can cause infections include skin, wound & blood,
and toxin related diseases
Cutaneous staph infections

folliculitis furuncle carbuncle

• Folliculitis infection of the hair follicles

• A furuncle is an acute, round, firm, tender, circumscribed,
perifollicular staphylococcal pyoderma that usually ends in central
suppuration.
• A carbuncle is two or more confluent furuncles with separate heads,
infection is deeper, may cause fever/chills
Bullous Impetigo
• Skin infection caused by
staph that is highly
infectious, by contact
with patient, fomites,
autoinnoculation
Staphylococcal toxin
related diseases
• Food poisoning
• Toxic shock syndrome
• Scalded skin syndrome
Staph food poisoning is an
enterotoxin
• Contaminated food kept at a
temperature that allows
bacterial growth and toxin
release
• Reheating the food may kill
the bacteria but the toxin is
heat stable
• Symptoms occur 1-6 hours
after ingestion, self limit at 24
hours
 Toxic shock syndrome
• Caused by the TSST-1 toxin 1
• Rare potentially fatal multi-system
disease with sudden onset of fever,
chills, vomiting, diarrhea, muscle
aches and desquamating rash that
can lead to hypotension and shock
• Originally described in 1978,
associated with highly absorbent
tampon use
• Localized infection, systemic toxin
Scalded skin syndrome
• More common in infants
• Epidermolytic toxin has widespread
effect
• Also known as Ritter disease
• Lesion has purulent material, then
cutaneous erythema followed by
peeling of the epidermis
• Lasts 2-4 days, spontaneously heals
Because of their thick cell wall,
Gram positive bacteria are often
treated with cell wall inhibitors

These are the beta lactams and

vancomycin
Targets and mechanisms of
action
 Beta lactam antibiotics
• Naturally occurring
• Synthetic
• Semi-synthetic
 Peptidoglycan structure of cell wall
Normal cell wall composition is alternating NAG and
NAM

Transglycosylator
enzymes

Crosslinking by
transpeptidation

Cell wall helps organism resist changes in osmotic conditions

Inhibition of cell wall
synthesis
• Beta lactams are structural
analogs to acyl D-alanyl-D-
alanine, and inhibit the
transpeptidation reactions.
• Penicillins
• Cephalosporins
• Monobactams
• Carbapenems
• Vancomycin (glycopeptide) is
narrow spectrum, limited to
gram positive. Cannot penetrate
the outer cell membrane of
gram neg
• Links to peptide substrate to
prevent transpeptidation
Summary: The beta
lactams interfere with cell
wall synthesis
• They look like the next building block for cell wall
synthesis
• If they are inserted by the Penicillin binding
proteins into the cell wall, cell wall synthesis will
stop
• Beta lactams include all penicillins, all cephems,
monobactams and aztreonams
Koneman 6th edition color atlas
MRSA Methicillin resistant S.
aureus
• MRSA is a major nosocomial infection, also called a
HA-MRSA. It can also be acquired in the
community, called CA-MRSA

• There are several strains and all of them have a

form of the SCCmec cassette, which is a large
insertion into the chromosome that brings several
genes, usually antibiotic resistance and virulence
genes, and all of them have the mecA gene
Penicillin Binding Proteins

• These are NORMAL components of a

bacterial cell well. They help complete
final transpeptidase reactions in cell
wall synthesis. They have been a drug
target, historically.

• mecA and mecC (heavy sigh) genes

• These genes change the PBP.

• PBP2a is a modified PBP with a lower

binding affinity for beta-lactamases,
rendering the abx less effective

• They may also see a change in their

upstream promoter regions so they
are over-produced to combat the
effects of beta lactams
Beta-lactamases
So, what do we do?
• We use those synthetic and semi-synthetic forms of
beta lactams to try to work around these defenses.

• For example, if the organisms is resistant to

penicillin, we may treat with nafcillin or oxacillin

• However, in MRSA (when we call an organism

“methicillin resistant,” we are saying it is resistant
to all beta-lactams
Fun fact! Microbes are
smart
• They have developed a fun new type of enzyme
called an extended-spectrum beta-lactamase

• We will revisit this in the Enterobacteriaceae, which

production of this type of enzyme is more common
 Testing for MRSA
• The drug oxacillin is used to detect methicillin
resistance. A plate with high salt and
polymixin make the media select for
Staphylococcus. Then, we drop an oxacillin disc

• CLSI recommends using cefoxitin plates to

induce the mecA gene because MR can show
heteroresistance

• PCR and real time PCR can also be used

• Surveillance cultures may be done on a target

population at high risk, eg. Nursing home
patients
Inducing Resistance…
• This bring us to constitutive or inducible resistance

• Constitutive – the gene for the resistance mechanism

is always on, ever vigilant

• Inducible – the gene is turned on in the presence of

the abx

• Bacteria have means for both when looking at beta-

lactamases, specifically
Vancomycin resistant S.
aureus
• Vancomycin interferes with cell wall synthesis but is
not a beta lactam

• VISA: Vancomycin intermediate S. aureus can arise

from vancomycin overuse

• VRSA: resistant S.A.

• Screening can use a vancomycin agar plate

Macrolide resistance in S.
aureus (because resistance to one WHOLE CLASS of antimicrobials isn’t enough)

• Macrolide antibiotics work by inhibiting protein synthesis at

the level of the ribosome
• Clindamycin and erythromycin are two examples, and
usually have the same susceptibility pattern. When they
don’t, it may be that the organism can be induced to be
resistant once the patient starts to take the antibiotic

E CC
Inducible Macrolide
resistance
• The D test places clindamycin and erythromycin in close
proximity to each other on a Kirby Bauer plate.

• The erythromycin makes the bacteria around it express its

erm gene, which makes it demonstrate its resistance to the
clindamycin
• The D test determines in advance if the organism will
respond to clindamycin
• If the D test is positive, report all
macrolides as resistant
S. Aureus Kirby Bauer

1 2

8
9
4

7
5

6
Add Results Here
Number Abx Used S or R
1 Gentamaycin (aminoglycoside, 30S)
2 Oxacillin (beta-lactam, cell wall)
3 Cefuroxime (cephalosporin, cell wall)
4 Pefloxacine (quinolone, DNA synthesis)
5 Clindamycin (macrolide, ribosome)
6 Erythromycin (macrolide, ribosome)
7 SXT (folic acid metabolism)
8 Vancomycin (cell wall)
9 Cefoxitin (beta-lactam, MRSA screen)
Virulence factors of
Staph aureus

• Enterotoxins
• TSST-1
• Exfoliative toxin
• Cytolytic Toxin
• alpha toxin
• Beta toxin
• Panton Valentine
leucocidin
• Delta toxin
• Enzymes
• Protein A binds Fc portion
of antibody, blocks
phagocytosis
Identification of S. aureus
from skin/wound infection
• Gram positive cocci in clusters

• Smooth white/yellow creamy colonies with beta

hemolysis on blood agar

• Catalase positive

• Coagulase positive

• Will this grow on a MAC? How about a CHOC?

S. aureus Identification
(GS/BAP)
Catalase Testing
Coagulase Testing
• Coagulase can present in bacteria as “free” coagulase,
“bound” coagulase or they can have both.

• Bound coagulase – attached to the bacterial cell. Heat

stable enzyme (sometimes called “clumping factor”)

• Free coagulase – secreted into the environment. Heat

labile.

• Serves a role in protecting the microbe from the immune

system
Coagulase test is positive
for S. aureus – bound and
free (+)
 pos
Gram _____Cocci
________
catalase Positive

coagulase
Positive

1 2

VP (+) VP (-)
Mannitol (+) Mannitol (-)

Vogues Proskauer/Mannitol to distinguish, only if the wound is from a dog bite

 Staphylococcus
intermedius
• Species name reflects how it is sort of an “intermediary”
between biochem results in S. aureus and S. epidermidis

• Coagulase result:
• Bound coagulase (slide) 50% (+)
• Free coagulase (tube) ~100%

• It’s in the cases it is positive, we want to R/O S. aureus

• We can do this with VP and Mannitol fermentation
 Staphylococcus epidermidis
• Commensal
• Found on skin
• Common
contaminant of
wound, blood
cultures

• Pathogen
• Slime / biofilm
• Endocarditis
• Nosocomial UTI
S. epidermidis – relevant
testing
• Catalase (+)

• Coagulase (-)

• Bacitracin (R) and Novobiocin (S) Susceptibilities

 Staphylococcus
saprophyticus
 saprophyte An organism that grows on and derives its
nourishment from dead or decaying organic matter.

 Urines – relevant sample type

 S. epidermidis – relevant
testing
• Catalase (+)

• Coagulase (-)

• Bacitracin (R) and Novobiocin (R) Susceptibilities

Mannitol Salt Agar
Differential Component:

Mannitol

pH Indicator:

Phenol Red
MSA in staphylococci
• Positive:
• S. aureus
• S. saprophyticus

• Negative:
• S. epidermidis
• S. intermedius
• S. lugdunensis
• M. luteus

Draw and label these results

Staphylococcus
lugdunensis
• Coagulase negative
• Can sometimes be slide positive, but always tube negative

• May contain the mecA gene

• Can cause infective endocarditis, septicemia, meningitis,

skin and soft tissue infections, UTIs and septic shock

• Has been confused with S. aureus. Can be identified as

it is PYR positive and ornithine decarboxylase positive
 Micrococcus
• Microdase (oxidase) +
• Nonfermenter (staph Staphylococcus
ferment)
• May be asacchrolytic
A
• Susceptible to lysozyme
• Resistant to lysostaphin
• Sensitive to bacitracin (A
disk)
A
• Resistant to furazolidone

Micrococcus
Stomatococcus
mucilaginous
• Medium white colony
• Strong adherence to agar – sticky
• Nonhemolytic
• Transmission same as Micrococcus, seen in bone
marrow recipients, malignancies
• Infection is rare
• Virulence is low
• Microdase disk negative [Micrococcus is positive]
 Gram _____Cocci
________ Positive
Positive coagulase

1 2
Negative

Bacitracin
VP+ VP----
3-
Sensitive Resistant

Novobiocin

Sensitive Resistant
May need to do oxidase
for (-) if
7
5. _______________ 6