MOD9 Staphylococcus

number of species

27 (25-27)

normal inhabitants of skin and mucous membrane of humans and animals

groups based on coag enzyme production

coagulase positive

coagulase negative

coagulase positive Staphylococcus

Staphylococcus aureus most common

coagulase negative Staphylococci CNS CoNS

Staphylococcus epidermidis, Staphylococcus hominis — most common

Staphylococcus saprophyticus — urinary pathogens (only in urine cultures)

Staphylococcus lugdunensis 

emerging pathogenic CNS

Gram stain and colonial morphology same as all other CNS

fools MLTs bcs it has bound coagulase, not free coagulase

slide coag+

tube coag-

Staphylococcus schleiferi subspecies

S.schleiferi subsp coagulans

S.schleiferi subsp schleiferi

S.schleiferi subsp coagulans

found mostly in dogs, can cause human infc

tests slide coag-

tests tube coag+

S.schleiferi subsp schleiferi

causes infc in humsns

tests slide coag+

tests tube coag-

emerging pathogenic CNS

Staphylococcus celluar morphology

Gram positive cocci

spherical — never oval

ave. 1um

clusters and tetrads — can appear in pairs if tetrads broken up

report as GPC STA

Staphylococcus growth requirements

facultative anaerobes — grow better in air

mesophilic — 35-37deg

non-fastidious — grow easily on most media

atrichous/non-motile

S.aureus colonial morphology BAP

• 1-4mm — generally larger than CNS colonies incubated for same time

• opaque — vanilla pudding-like

• b-hemolytic, occasionally non-hemolytic — can also see double zone of hemolysis

• gold “aura”

• creamy yellow colour — lipochrome pigment (can take 48-72hr)

mannitol salt agar MSA

selective and differential media for S.aureus

rapid detection by inhibition of flora

selective property for Staphylococcus spp

differential property for S.aureus

MSA selective property

selective for Staphylococcus spp

high tolerance for salt, can grow on media w 7.5% NaCl

other organisms inhibited

MSA differential property

differentiates mannitol fermenters vs non fermenters

contains carb mannitol and pH indicator phenol red

fermentation of mannitol → acid production = yellow colour

S.aureus ferments, CNS does not

MSA uses

not routinely used for detection bcs incubation time

use vitek or maldi

used to screen healthcare workers/pts for carriage

latex kits

particles w fibrinogen attached and IgG Abs

specific protein A on S.aureus cells bins to Fc portion of IgG → visible agglutination

more accurate than slide coagulase

hemagglutination test 

reagent: RBCs w fibrinogen attached

bound coagulase + reagent: immediate clumping of RBCs

thermostable nuclease test

heat stable nuclease enzyme uniquely produced by S.aureus

used in food mbio — food poisoning cases

what tests do CNS test positive for

catalase

nitrate reduction

4% of DNase testing

what test can give a negative result with S.aureus 

DNase — occasional negative

S.aureus pathogenicity

most common cause of skin, soft tissue and post traumatic infc, joint infc, bacteriemia

always a primary pathogen

susceptibility performed upon isolation — exception: known sites ex. nose

S.aureus virulence factors 

• leukocidin

• hyaluronidase

• staphylokinase

• hemolysins

• DNase

• coagulase

• beta-lactamase

• toxic shock syndrome toxin-1 TSST-1 — superantigen, causes overactive immune response

• protein A — binds IgG and prevents phagocytosis 

• enterotoxins — food poisonings

• exfoliative toxins

• lipase — degrades lipids on skin surface → greater susceptibility 

furuncle/boil

localized abscess around hair follicle

carbuncle

cluster of boils that form a connected area of infc under the skin

stye

bacterial infc involving one or more small glands near base of eyelashes

impetigo 

superficial skin infc, water blisters on the face and body

absecess

deep soft tissue infc

wound infc

bacterium penetrate deeper tissue after break in skin

osteosyelitis 

superficial infc that spreads via blood to bone 

septicemia

localized infc w bacteria spreading and proliferating in blood

heat stable enterotoxin

affects intestinal cells

preformed toxins in food → vomiting diarrhea 1-6hr post ingestion

moist, protein food contamination

toxic shock syndrome toxin-1 TSST-1

causes toxic shock syndrome TSS

S.aureus grows on tampon and liberates toxin internally

absorbed and spread by blood stream

symptoms: high fever, sore throat, nausea, vomiting, diarrhea, sunburn-like rash

death can occur: hypotension, resp and cardiac distress, renal failure

exfoliative/epidermolytic exotoxins A and B 

causes staphylococcal scalded skin syndrome SSSS (aka ritter disease)

produced by certain strains of S.aureus

common in children and immunosuppressed 

toxin at site of boil or skin infc spreads to other areas, exposes dermis giving glistening appearance

methicillin resistant staphylococcus aureus MRSA

20-30% S.aureus are MRSA

increased mortality — varies with site of infc, age of pt, comorbidities, etc

resistant to vast majority of beta lactams

drug of choice: vancomysin

kinds of MRSA

hospital/healthcare acquired — HA-MRSA

community acquired — CA-MRSA

what happens to pts who are MRSA positive 

put in isolation, caretakers don additional precautions — masks, disposable gowns

cohorting — placed with other MRSA positive pts

MRSA admission screening sites

areas known to have high colonization rates (nares, inguinal) swabbed and plated on selective chromogenic media (denim blue agar DBA)

colonies will show up as blue — allows for rapid detection and isolation

selective chromogenic media

good for isolating colonies from sites heavily colonized by normal flora

where is Staphylococcus found

non-sterile: wounds, drainages, abscess

sterile: blood, synovial fluid, tissues

MRSA susceptibility pattern — vitek card

cefoxitin screen — pos

benzylpenicillin — R

oxacillin — R

vancomycin — S

MRSA lateral flow test

definitive test

targets PBP2A 

result in 5 mins

has internal QC

advantage: allows for early detection and tx prior to having suscept. results

oxacillin screen plate

oxacillin diluted into agar (agar dilution method)

inoculum deposited onto agar

growth → org can grow in presence of abx and high [NaCl] = pos MRSA

no growth → isolate is susceptible to oxacillin therefore MSSA

mecA gene detection

polymerase chain reaction PCR detection of mecA gene therefore resistant

what needs to be determined if theres a MRSA outbreak 

1. source of outbreak — patient information, site of infc (nose, inguinal, rectal)

2. are all isolates implicated in the outbreak the same strain 

MRSA fingerprinting

determine whether outbreak is caused by single or multiple strains

1. bacteriophage testing

2. pulse-field gel electrophoresis PFGE

bacteriophage testing 

specific phage for specific strain of bacterium

expose suspect bacterium to a selection of phages

bacteriophage

virus that infects bacterial cells

pulse-field gel electrophoresis PFGE

bacterial DNA is cleaved into fragments → separated on a gel

gel strip is stained and results are compared to known markers

what type of pathogen are CNS

opportunistic pathogens 

where is CNS found

large portion of normal flora of skin and mucous membranes — nose, genitals

CNS colonial morphology

1-2mm in size

opaque

usually dull, white, non hemolytic

never alpha hemolytic

different between S.aureus and CNS on a BAP 

S.aureus: larger, b-hemol, yellow

CNS: smaller, non-hemol, white

CNS identification methods

biochemical testing

Gram stain = GPC STA → catalase+ → slide/tube coag-

commercial systems

vitek, maldi — always supplemented with some minimal biochem testing

CNS susceptibility

more resistant than S.aureus but much less likely to cause infections

penicillin sensitive but methicillin resistant

Staphylococcus saprophyticus pathogenicity 

CNS only considered a pathogen when found in significant colony count in urine cultures 

Staphylococcus saprophyticus colonial morphology

slightly larger than other CNS

shiny, smooth/buttery

bright white, can be yellow or orange

non-hemolytic

novobiocin disk

Staphylococcus saprophyticus identification

only spp resistant to novobiocin

zone size less than/equal to 12mm is diagnostic

other CNS will have susceptible zone size of greater than 12mm

only used for identification, not tx

which spp is resistant to novobiocin 

Staphylococcus saprophyticus

novobiocin test procedure

done on BAP or MH

standard inoculum — 0.5mcfarland for BAP, 1.0 for MH

streak plate for confluent growth, add disk

incubate overnight in O2

Micrococcus spp

normal flora of skin, mucous membranes, oropharynx

GPC STA

colonial morphology:

• at 24hr: small, white, non-hemolytic

• at 48hr: 1-2mm, commonly bright yellow, can also be white/tan/orange/pink/lime green

catalase+, not considered CNS

rarely considered pathogenic

bacitracin disk

Micrococcus spp identification

susceptible zone size of greater than/equal to 10mm

all other cat+ GPC will be resistant

Kocuria spp pathogenicity 

non-pathogenic 

Aerococcus urinae

associated with urine cultures

cause invasive conditions — endocarditis

GPC STA

differentiating features from Staphylococcus spp:

• alpha-hemolytic

• catalase neg

Aerococcus urinae susceptibility

sensitive to penicillin, cephalosporins, vancomycin, nitrofurantoin

resistant to SXT, trimethoprim, ciprofloxacin

use maldi for identification 

Staphylococcus identification flowchart

Gram stain = GPC STA

catalase = positive

(nitrate = positive after addition of reagents A and B)

slide coag =

• negative: CNS → tube coag negative

• positive: Staphylococcus aureus → tube coag also positive

urine specimens:

CNS → novobiocin disc

resistant: less/equal 12mm → Staphylococcus saprophyticus

susceptible: greater than 12mm → other CNS (ex. Staphylococcus epidermis)

what isolates could result in slide coag+ and tube coag-

Staphylococcus lugdunensis

Staphylococcus schleiferi subsp schleiferi

what isolates could result in slide coag- and tube coag+

Staphylococcus schleiferi subsp coagulans

infections caused by CNS

bacteriemia, septicemia, meningitis, UTI