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MBIO345 MB > Module 9 > Flashcards

Module 9 Flashcards

1
Q

What family does Staphylococcus belong to

A

Micrococcaceae

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2
Q

The genus Staphylococcus is divided into : (2)

A

Coagulase positive Staphylococcus

Coagulase negative Staphylococcus

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3
Q

Coagulase positive Staph

A

S. aureus most common
S. schleiferi subspeicies coagulans
S. hyicus (coagulase variable) and S. intermedius are isolated from animals

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4
Q

Coagulase negative Staph

A

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”

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5
Q

Cellular morphology of Staphylococcus

A

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)

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6
Q

Staphylococcus growth requirements

A

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

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7
Q

Colonial morphology of Staphylococcus

A

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)

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8
Q

3 types of hemolysis

A

Beta
Alpha
Gamma

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9
Q

Beta hemolysis

A

complete clearing of RBC around colony

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10
Q

Alpha hemolysis

A

partial clearing of RBC

Greening around colony

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11
Q

Gamma hemolysis

A

No clearing of RBC around colony

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12
Q

Lipochrome

A

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

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13
Q

Why can’t pigmentation be a reliable feature for identification

A

some strains are not pigmented (genetic control)
Anaerobic incubation inhibits pigmentation
Overnight colonies often not pigmented

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14
Q

Are CNS colonies pigemented

A

No

Usually white

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15
Q

Can Staphylococci tolerate high levels of salt

A

yes

Salt is used to make selective media for isolating Staphylococci

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16
Q

Mannitol salt agar (MSA)

A

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

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17
Q

MSA growth interpretation

A

Growth on MSA = likely Staphylococcus
Mannitol negative colonies (red) = likely CNS
Mannitol positive colonies (yellow) = Likely S. aureus; a few CNS do ferment mannitol

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18
Q

cons to using MSA plates

A

$$$$
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

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19
Q

Genus Identification of Staphylococcus (2)

A 
Catalase = pos with fast production of bubbles
Nitrate = pos with nitrate reduced to nitrite (red color after addition of reagent A and B)
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20
Q

If slide coagulase is positive,

A

organism may be identified as S. aureus with confidence

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21
Q

If slide coagulase is negative,

A

often a tube coagulase will be set up

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22
Q

Hemagglutination test for bound coagulase

A

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

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23
Q

Latex reagents for ID of S. aureus

A

Reagent of latex particles with attached fibrinogen and IgG antibodies
Reagent will react with clumping factor (fibrinogen) and protein A (IgG) to produce agglutination

24
Q

Protein A

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

25
Q

Why is latex test considered more accurate than slide agglutination for testing S. aureus

A

latex detects both protein A and clumping factor

26
Q

DNase test for S. aureus

A

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

27
Q

Thermostable Nuclease Test and S. aureus

A

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)

28
Q

CNS saprophyticus colonial morphology

A

BAP: 1-2mm, gamma hemolysis, BRIGHT white, opaque, colonies appear wet, glistening and run together
MAC: no growth
Atrichous, no flagella

29
Q

What common infection does S. saprophyticus cause

A

UTI

only needs to be differentiated from other CNS when isolated in significant amounts from urine samples

30
Q

Test to differentiate S. saprophytic from other CNS

A

Novobiocin susceptibility test

**for ID not treatment testing

31
Q

Novobiocin susceptibility test

A

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

32
Q

When species ID is required for CNS

A

should be determined that isolate is actually a staphylococcus species and not Micrococcus
Do Bacitracin Disc test to determine

33
Q

Bacitracin disc testing

A

Similar procedure to Novobiocin susceptibility test but uses Bacitracin disc instead

Staphylococcus = resistant, no zone of inhibition
Micrococcus = susceptible, zone of inhibition
34
Q

Micrococcus species

A

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

35
Q

3 methods of determining actual species of CNS

A 
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!!!)
36
Q

Virulence factors of S. aureus (4)

A

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)

37
Q

Other Staphylococcal virulence factors (3)

A

Dnase, coagulase, beta-lactamase production

38
Q

Common S. aureus infections

A 
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
39
Q

Diseases caused by exotoxins

A

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)

40
Q

Where are CNS seen as normal flora

A

Skin
Nose
Genital mucosa

41
Q

Septicemia

A

bacteria have invaded the blood stream

often following infection of heart valves

42
Q

Meningitis

A

bacteria have invaded the spinal fluid and infected the meninges lining the brain

43
Q

S. aureus and Benzylpenicillin (pen G)

A

used to be universally susceptible
Now, 85-90% of S.aureus show resistance
Due to b-lactamase enzyme

44
Q

MRSA

A

20-30% of S. aureus are MRSA

Vancomycin only choice for treatment

45
Q

ID problems with MRSA

A

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

46
Q

Detection procedures

A

S. aureus can contain 2 subpopulations of bacteria
1 resistant to methicillin and the other not
“heteroresistant strains”
Resistant strain is slower growing

47
Q

Standardized inoculum for MRSA

A

use Direct method (take enough colonies to match McFarland standard)
This way you get both subpopulations, not just the fast growing one

48
Q

Incubation temp for MRSA

A

full 24hr incubation between 30-35degC

49
Q

Additional NaCl for MRSA

A

supplementing test medium with NaCl enhances growth of resistant populations
2% NaCl for broth but NOT MH agar for disc diffusion

50
Q

Disc diffusion testing for MRSA

A

Oxacillin discs
MRSA gives small zone of inhibition with feathering on the outside
Zone size is taken inside irregular growth

51
Q

MRSA oxacillin screen test (agar dilution test)

A

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

52
Q

New MRSA drugs

A 
Linezolid (Oxazolidinones class)
Quinupristin
Dalfopristin
Daptomycin
Tigecycline
53
Q

Antimicrobial susceptibility of CNS

A

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)

54
Q

Epidemiology

A

study of the sources and spread of infection

55
Q

Bacteriophage typing

A

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

56
Q

Pulse-field Gel Electrophoresis

A

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

57
Q

Mec A gene testing

A

Epidemiology research test
mec A gene codes for resistance to methicillin and other b-lactam antimicrobials
Molecular testing by PCR (polymerase chain reaction) testing

MBIO345 MB (13 decks)

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