Module 9 Flashcards
What family does Staphylococcus belong to
Micrococcaceae
The genus Staphylococcus is divided into : (2)
Coagulase positive Staphylococcus
Coagulase negative Staphylococcus
Coagulase positive Staph
S. aureus most common
S. schleiferi subspeicies coagulans
S. hyicus (coagulase variable) and S. intermedius are isolated from animals
Coagulase negative Staph
S. epidermidis most common
S. saprophyticus (cause of UTI)
S. lugdenensis (slide coagulase +, tube coagulase -)
Only S. saprophyticus is clinically significant
All reported as “coagulase neg Staphylococcus”
Cellular morphology of Staphylococcus
Gram pos cocci (spherical, NOT oval)
0.4-1.2um (average of 1.0um)
Typically in grape-like clusters but may also see singles, pairs, short chains (4-6 long)
NEVER in long chains (>6 cells long)
Staphylococcus growth requirements
Facultative anaerobes (can grow w or w/o O2 but prefer with)
Range 10-42 deg C (optimum 35-37)
Grows on most media; No growth on MacConkey
Motility - negative; atrichous
Colonial morphology of Staphylococcus
overnight colonies 1-4mm
S. aureus colonies tend to be larger than CNS
Opaque, dense, unable to see through “vanilla pudding”
S. aureus usually shows narrow zone of beta hemolysis
Some strains show double zone of hemolysis (extended incubation; inner zone complete hemolysis, outer zone partial hemolysis)
3 types of hemolysis
Beta
Alpha
Gamma
Beta hemolysis
complete clearing of RBC around colony
Alpha hemolysis
partial clearing of RBC
Greening around colony
Gamma hemolysis
No clearing of RBC around colony
Lipochrome
typical colonies of S. aureus produce a creamy-yellow color due to pigment Lipochrome after 48hr incubation
Lowering temp (room or fridge) after colonies have formed will enhance pigmentation
Why can’t pigmentation be a reliable feature for identification
some strains are not pigmented (genetic control)
Anaerobic incubation inhibits pigmentation
Overnight colonies often not pigmented
Are CNS colonies pigemented
No
Usually white
Can Staphylococci tolerate high levels of salt
yes
Salt is used to make selective media for isolating Staphylococci
Mannitol salt agar (MSA)
7.5% sodium chloride
Allows growth of Staphylococcus but inhibits most other bacteria
Differential medium due to presence of mannitol (carb) and pH indicator (phenol red)
Colonies that ferment mannitol produce acid causing indicator to turn yellow around colony
MSA growth interpretation
Growth on MSA = likely Staphylococcus
Mannitol negative colonies (red) = likely CNS
Mannitol positive colonies (yellow) = Likely S. aureus; a few CNS do ferment mannitol
cons to using MSA plates
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Requires 48hr incubation for mannitol fermentation to be detected
Colonies cannot be used directly from MSA for identification
Subculture is required = adds extra day to identification time
Genus Identification of Staphylococcus (2)
Catalase = pos with fast production of bubbles Nitrate = pos with nitrate reduced to nitrite (red color after addition of reagent A and B)
If slide coagulase is positive,
organism may be identified as S. aureus with confidence
If slide coagulase is negative,
often a tube coagulase will be set up
Hemagglutination test for bound coagulase
commercial kit for rapid id of S. aureus
Reagent of RBC sensitized with fibrinogen
Causes almost immediate clumping of RBC when reagent is mixed with bacteria on slide
Latex reagents for ID of S. aureus
Reagent of latex particles with attached fibrinogen and IgG antibodies
Reagent will react with clumping factor (fibrinogen) and protein A (IgG) to produce agglutination
Protein A
cell wall protein found in S. aureus (seldom in other Staphylococcus)
Binds to nonspecific IgG molecules by fragment crystallizable (Fc) portion of the antibody
IgG is bound to latex and leaves Fc portion exposed
If protein A is present, IgG and attached latex will bind to staph = agglutination
Why is latex test considered more accurate than slide agglutination for testing S. aureus
latex detects both protein A and clumping factor
DNase test for S. aureus
occasionally used to confirm ID of S. aureus but should not be used as sole identification test
Occasional strain of S. aureus is negative and 4% of CNS are DNase positive
Thermostable Nuclease Test and S. aureus
S. aureus produces heat stable nuclease enzyme that is not produced by other bacteria
Used mostly in food microbiology and food poisoning investigation
Has been adapted for direct ID from blood cultures (takes 2-3hrs, faster than waiting for colonies to form on media used for sub planting the broth)
CNS saprophyticus colonial morphology
BAP: 1-2mm, gamma hemolysis, BRIGHT white, opaque, colonies appear wet, glistening and run together
MAC: no growth
Atrichous, no flagella
What common infection does S. saprophyticus cause
UTI
only needs to be differentiated from other CNS when isolated in significant amounts from urine samples
Test to differentiate S. saprophytic from other CNS
Novobiocin susceptibility test
**for ID not treatment testing
Novobiocin susceptibility test
S. saprophyticus = resistant, zone size of = 12mm
Other CNS = susceptible (sensitive), >12mm
Organism grown in broth until it matches 0.5 McFarland standard.
Broth spread over surface of BAP or MH agar and 5ug novobiocin disc added
Incubate overnight and observe zone sizes
When species ID is required for CNS
should be determined that isolate is actually a staphylococcus species and not Micrococcus
Do Bacitracin Disc test to determine
Bacitracin disc testing
Similar procedure to Novobiocin susceptibility test but uses Bacitracin disc instead
Staphylococcus = resistant, no zone of inhibition Micrococcus = susceptible, zone of inhibition
Micrococcus species
normal flora of skin, mucous membrane and oropharynx region
BAP= small, non-hemolytic colonies (could be white, tan, bright yellow, orange, pink)
Colonies are catalase pos and rarely cause infections
3 methods of determining actual species of CNS
Conventional biochemical tests (take 2-3 days and requires range of tests not readily available) Commercial system (rehydrated with a suspension of bacteria and incubated from 5-24hrs) Automated Instrument (vitek provides gram pos ID; 4-24hr results; antibiotic susceptibility testing provided; Maldi-Tof ID in 2 hrs!!!)
Virulence factors of S. aureus (4)
Leucocidin (kills WBC)
Hyaluronidase (Breaks down hyaluronic acid in tissue and allows bacteria to spread)
Staphylokinase (lyses clot formed around site of infection; allows bacteria to spread)
Hemolysins (lyses RBC, weakens the host)
Other Staphylococcal virulence factors (3)
Dnase, coagulase, beta-lactamase production
Common S. aureus infections
furuncle: boil; localized abscess around hair follicle Carbuncle: several boils; deep tissue Stye: eye last infection Impetigo: superficial skin infection; watery blisters Abscesses: deep soft tissue infection Joint infection Infected cuts, abrasions, trauma wounds Infected incisions (2-3 days post op) Pneumonia Osteomyelitis Septicemia ****ANYTHING CAN CAUSE THIS
Diseases caused by exotoxins
Food poisoning (certain heat stable S. aureus; upper/lower shelf issues 1-6hrs post consumption)
Toxic Shock Syndrome (S.aureus grows on tampon; high fever, sore throat, upper/lower shelf issues, sunburn-like rash, hypotension, respiratory/cardiac distress, renal failure)
Scalded skin Syndrome (sunburn like rash, glistening skin, red dermis exposed)
Where are CNS seen as normal flora
Skin
Nose
Genital mucosa
Septicemia
bacteria have invaded the blood stream
often following infection of heart valves
Meningitis
bacteria have invaded the spinal fluid and infected the meninges lining the brain
S. aureus and Benzylpenicillin (pen G)
used to be universally susceptible
Now, 85-90% of S.aureus show resistance
Due to b-lactamase enzyme
MRSA
20-30% of S. aureus are MRSA
Vancomycin only choice for treatment
ID problems with MRSA
some strains are slow growing
produce small colonies after 24hrs incubation that resembles CNS colonies (less than 1mm and white)
May give neg slide coagulase and require full 24hr incubation to give a positive tube coagulase test
These may be considered normal CNS flora when only a bound coagulase test is used to screen for S. aureus
IDEALLY: All slide CNS should have a tube coagulase done with full 24hr incubation
Detection procedures
S. aureus can contain 2 subpopulations of bacteria
1 resistant to methicillin and the other not
“heteroresistant strains”
Resistant strain is slower growing
Standardized inoculum for MRSA
use Direct method (take enough colonies to match McFarland standard)
This way you get both subpopulations, not just the fast growing one
Incubation temp for MRSA
full 24hr incubation between 30-35degC
Additional NaCl for MRSA
supplementing test medium with NaCl enhances growth of resistant populations
2% NaCl for broth but NOT MH agar for disc diffusion
Disc diffusion testing for MRSA
Oxacillin discs
MRSA gives small zone of inhibition with feathering on the outside
Zone size is taken inside irregular growth
MRSA oxacillin screen test (agar dilution test)
MH agar with 6ug oxacillin/ml and 4% NaCl added
0.5 McFarland standard is spot inoculated to the plate
After incubation (24hr 35degC), any growth = resistance
New MRSA drugs
Linezolid (Oxazolidinones class) Quinupristin Dalfopristin Daptomycin Tigecycline
Antimicrobial susceptibility of CNS
CNS generally more resistant than S. aureus with exception of MRSA
S. saprophyticus not as resistant as other CNS and is susceptible to most antimicrobials. Quinolones often used (norfloxacin, Nitrofurantoin)
Epidemiology
study of the sources and spread of infection
Bacteriophage typing
Epidemiology research test
bacteriophages are viruses that infect bacterial cells
Phages will lyse host bacteria resulting in plaques or clear areas in bacterial growth
Host specific
Pulse-field Gel Electrophoresis
Epidemiology research test
uses DNA technology to cleave sections of bacterial chromosome into smaller pieces
DNA fragments are separated by gel medium and electrical current
Mec A gene testing
Epidemiology research test
mec A gene codes for resistance to methicillin and other b-lactam antimicrobials
Molecular testing by PCR (polymerase chain reaction) testing
