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Micro240 > GRAM-POSITIVE AST review > Flashcards

GRAM-POSITIVE AST review Flashcards

1
Q

Staphylococcus aureus

A

-NF in skin or nose - can cause infection -makes virulence factors
-first treated with Penicillin - beta lactam AB
*Beta lactam ring of antibiotic binds to PBP binding site prevents bacterial cell wall synthesis = cell death

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2
Q

S. aureus BETA LACTAMASE PRODUCTION

A

-when penicillin is over used then S aureus will take blaZ gene from plasmid
* the gene Codes for production of serine -lactamase enzyme β called penicillinase
-* penicillinase Destroys -lactam ring β of antibiotic & inactivates it.
*Enzyme is inducible- made when organism exposed to penicillin

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3
Q

CONFIRMING A PENICILLIN SUSCEPTIBLE S. aureus

A

-90-95% of S. aureus make penicillinase Beta lactamase.
-VITEK puts staph as B lactamase pos regardless of what the Pen MIC says meaning vitek will call s aureas PEN R
-always check benzylpenicillin MIC or KB zone result before reporting as PEN R.
*If the S. aureus has a susceptible penicillin MIC (≤0.12 μg/ml) or disk zone (≥29 mm), must perform a Beta lactamase test to determine if should really be susceptible.

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4
Q

BETA LACTAMASE TESTING FOR S. aureus

A

*If only 1 test do Penicillin zone edge test - more reliable than nitrocefin - lactamase test.β
*If doing >1 test start with nitrocefin (result in an hour) , if pos report as BL pos
*If negative (remember wait 1 hour before calling it negative) perform Pen zone edge test to confirm.

0.5mCF of staph on MH plate on lawn of growth - O2 35

*Fuzzy beach edge = BL neg & Pen S
*Sharp cliff edge= BL pos & Pen R

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5
Q

WHAT DOES BL POS MEAN IN S. aureus

A

*Penicillinase made by S. aureus only affects penicillinase sensitive antibiotics like: Penicillin, ampicillin, amoxicillin,, carbenicillin, mezlocillin, piperacillin, and ticarcillin.
*Doesn’t act on penicillinase R antibiotics like oxacillin & other

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6
Q

What are some AB with beta lactam ring

A

*Penicillin, ampicillin, piperacillin
*1st, 2nd, 3rd generations of cephalosporins
*Monobactam (Aztreonam)
*Carbapenems (Imipenem, meropenem, ertapenem)

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7
Q

WHAT IS MRSA?

A

Penicillinase resistant antibiotics:*methicillin & nafcillin (NOT USED because unstable).
*Oxacillin, cloxacillin & dicloxacillin–(in use because more stable/reliable results)
-staph acquires mecA or mec C gene carried on a SCC mec
-Codes for binding site modification of PBP2 to PBP2a or PBP2’
*Penicillinase resistant antibiotic not inactivated -just can’t attach to target site (PBP2) anymore
-the staph strains with these genes are called MRSA

Resistance due to target site modification
An MRSA, is resistant to:
*All penicillins including those that are penicillinase R
*Cephalosporins
-Carbapenems

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8
Q

CLSI SCREENING/TESTING RECOMMENDATIONS FOR MRSA

A

1.Oxacillin MIC & cefoxitin screen (Vitek AST) or a KB with a 30ug Cefoxitin disk (test with cefoxitin disc, but report oxacillin)

2.Oxacillin screen plates: MHA 6 g/ml of oxacillin & 4% NaCl.

Possible MRSA indicated by:
*Oxacillin with a R MIC + cefoxitin screen positive on Vitek
*30ug cefoxitin disk is R by KB
*And the S. aureus grows on the OX screen test plate

3.Confirm possible MRSA by latex agglutination, immunochromatographic membrane test (test for PBP2a) or PCR for mec A or C gene.

4.Vancomycin Screen to detect VISA or VRSA

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9
Q

OXACILLIN R DETECTION ISSUES WITH SOME MRSA *******

A

*Any MRSA with inducible OX R might test as cefoxitin screen pos or R, but Oxacillin S. so if you tested with OX you would call MRSA and MSSA.
-Inducible R happens with mecA but not often
-mostly happens with mecC because the mecC PBP2A has a higher affinity for ox but is R to cefox

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10
Q

Why is Cefoxitin is better predictor of MRSA

A

*More reproducible
*Better at inducing the organism to alter its PBP2 to PBP2

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11
Q

problems with detecting mec C MRSA

A

*PBP2a latex aggl tests give false-negative results for mec C.
*PCR assay typically test for mec A gene, need one with also mec C-specific primers

In most mecC MRSA, OX tests as S or Ion Vitek but cefoxitin screenwill be positive/R.
*Can also happen with mec AMRSA ,it happens more often with mec C.

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12
Q

HETERORESISTANCE IN MRSA

A

mix of OX R & OX S, S. aureus in a single population of isolate
-all have the gene for OX R but not many express it so you may miss it
-seen by some colonies from pure culture growing in the zone of inhibition

-be sure to pick up multiple colonies
-R strains are slower growing , incubate MRSA for full 24 hours so you can see slow growing resistant strains

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13
Q

BORSA

and signs of borsa

A

Staph that hyperproduce BL which is why there are borderline resistant to OX
-MICs are just above the breakpoint for OX sus
-Borderline Oxacillin Resistant strains of S. aureus
-dont have mecA or mecC gene – not MDR
-susceptible to cefoxitin
-Borderline resistance to Ox can also be due to an altered PBP2 but NOT PBP2A the one associated with MRSA

Signs of a possible BORSA:
*May be oxacillin R on Vitek because it has the normal PBP and makes alot of BL
*Cefoxitin screen neg or S
*May not grow on 6ug/ml oxacillin screen plate but can be still resistant but can be picked up on the Vitek to pick those with a lower MIC

BORSA usually around 1-8 µg ml
·MRSA is usually > or equal to 8
CLSI cut off for OX R is > =4

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14
Q

how to do the OXACILLIN SCREEN FOR MRSA

A

Media:
2 Mueller Hinton agar plates (control & test) Growth Control plate: MHA + 4% NaCl
Test plate: MHA + 4 % NaCl + 6ug/ml of oxacillin

*NaCl added to media since S. aureus is halophilic (salt loving)
*Test with oxacillin because methicillin in unstable & results unreliable Inoculum:
*Make 0.5 Mcfld of organism to be tested.

*CLSI recommends using 1- L loop, or a swab dipped in μthe suspension & with excess fluid expressed to spot area 10–15 mm diameter on each plate.

Incubate for a full 24 hours at 350C, O2.Temperature must not exceed 350C

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15
Q

OXACILLIN SCREEN LIMITATIONS **

A

-use multiple colonies for 0.5 McF
1 uL loop to inoculate plates - improves detection of R, in heterogeneous resistant population.
*Incubate full 24hrs in O2 35 - MRSA are slower growers
*All organisms must grow on MH w salt and QC organisms must perform as expected to report results
*Inspect carefully -even one colony on oxacillin plate, may indicate an MRSA
Confirm possible MRSA by testing for PBP2a or (Mec A or C gene) by PCR
.OX screen is not used to test for MRSA directly from clinical specimens – only done from growing colonies.
*Does not detect all BORSA
*Not used to detect OX R CONs (won’t grow on agar w salt)

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16
Q

30ug CEFOXITIN DISC TEST

A

-KB w 30ug cefoxitin disk is an alternative to see if there is Oxacillin R in staph
-cefoxitin gives more reproducible and accurate results than OX
-OX isnt reliable because you can only see the resistance after inducing
*Cefoxitin is better inducer of PBP2a/2’ in mec A or C strains
-surrogate for oxacillin -test using cefoxitin but report oxacillin
-can test for OX R in some other Staphylococcus spp. including S. lugdunensis.

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17
Q

β-lactamase negative and susceptible to cefoxitin

interpret

A

Susceptible to Penicillins Susceptible to penicillinase-resistant penicillins (e.g., oxacillin

18
Q

β-lactamase positive and susceptible to cefoxitin

interpret

A

Resistant to penicillin; Susceptible to penicillin-beta-lactamase inhibitor combinations and penicillinase-resistant penicillins (e.g., oxacillin)

19
Q

S. LUGDUNENSIS AST CONSIDERATIONS

A

-test by VITEK GP AST or KB
-able to acquire virulence factors
-can be Ox resistance due to mecA - rare
*BL test not needed, if BL pos is always PEN R by MIC/KB
-same OX & cefoxitin breakpoints as S. aureus

-report Ox result as S or R from Vitek OX MIC result or KB, or by using 30ug cefoxitin disk–(test with cefoxitin report oxacillin)
-no OX screen - wont grow some lug cant grow in salt media

-no need to do PBP2a or PCR to confirm OX R.
- to Vancomycin so Vanc screen not done

not of IC concern

19
Q

Resistant to cefoxitin(will automatically be considered β-lactamase positive

interpret

A

Resistant to penicillin, penicillin-beta-lactamase inhibitor combination and penicillinase-resistant penicillins like oxacillin as well as carbapenems

20
Q

VANCOMYCIN RESISTANT ENTEROCOCCUS

A

-NF in intestine, wound, urine
-opportunistic
-*Intrinsically R to penicillin & cephalosporins – some strains R to low levels vancomycin
*Acquired R to erythromycin, tetracycline, & high-level aminoglycosides. and .now vancomycin.
*Vancomycin kills organism by preventing cell wall synthesis – drug of last resort to treat MDR Enterococci
-Van A & Van B are most clinically relevant for vancomycin resistance
*Enterococcus faecium & Enterococcus faecalis acquire Van A or Van B via transposons to become Vanc R.
*Enterococcus gallinarum and cassiloflavus (& others) intrinsically (naturally) low level R to Vanc –due to Van C

21
Q

WHY ITS IMPORTANT TO SPECIATE ENTEROCOCCUS

A

-Species level ID confirms if intrinsic (Van C) or acquired (Van A or Van B
-need for infection control
Van C genes are intrinsic & not transferable, do not often cause serious infections, & not associated with outbreaks
.Van A and Van B genes are acquired & transferable can spread & associated with outbreak

22
Q

RELATIONSHIP BETWEEN VRE & MRSA VISA/VRSA

A

-Vancomycin was used to treat staph mostly MRSA
-increased use caused spread of Vancomycin resistant Enterococcus(VRE).
* VRE spread plasmid-borne resistance genetic element to S. aureus.
*Resulted in emergence of vancomycin intermediate S. aureus (VISA) or vancomycin resistant S. aureus (VRSA).
*Resistance to vancomycin leaves fewer treatment options

23
Q

VISA/VRSA TESTING

A

-set up Vanc screen on MRSA to see if VISA/VRSA
-screen plates will only detect vancomycin I or vancomycin R if the organism has an MIC of 8ug/mL or greater
*VISA have a Vanc MIC from 4 - 8ug/ml
*VRSA have Vanc MIC of ≥16ug/ml.
*Means all VRSA would be detected but some VISA would not be detected by Vanc Screen which is why we need the Vanc MIC from VITEK or you can get the MIC from doing Van E test
-can also send to PHL to confirm

24
Q

VANCOMYCIN SCREEN for VRE

how is it done

A

Media:Use 2 BHI agar (control & test)
Control plate: BHI agar
Test plate: BHI agar + 6ug/ml vancomycin
Inoculum:*0.5 Mcfld standard
*spot of the control plate and test plate with swab

Quality Control: VRE(E. faecalis R strain) & VSE(E. faecalis S strain) on both the growth control & test BHI plates.

Incubation: A full 24 hours at 350CO2.

Results: Any growth on plate with vanc indicates possible VRE. Organism must also show growth on the control plate & QC organisms must perform as expected.

25
Q

VANCOMYCIN SCREEN LIMITATIONS

A

*All organisms must grow on BHI without Vanc and QC
*Screen used only for Enterococcus & MRSA.
*Some enterococci are intrinsically R to vanc so any showing vanc R should be speciated
*any E. faecalis or faecium, that are a possible VRE must be tested with MIC method determine exact concentration of vanc it is R to.
*PCR to determine which gene VanA, VanB or VanC is present can be done for IC purposes.

26
Q

ENTEROCOCCUS –STERILE SITES REQUIRED AST TESTING

A

For all sterile sites, ID must be to species level:
1.Do gram, cat, PYR
2.MALDI ID

AST testing involves: - sterile sites
3.Vitek GP AST
4.VANC Screen
5.High Level Aminoglycoside Testing (HLAR)

27
Q

ENTEROCOCCUS AST CONSIDERATIONS

A

-ampicllin for non serious infections
*Can become R to amp by slightly altering PBP -antibiotic can’t bind as strongly
*Organism not killed by antibiotic- but growth inhibited
-amp still can be used because the slow growth will let immune system fight off infection
-sometimes Enterococci can completely alter their PBPs & become completely R to ampicillin you can use Vanc to treat
-rarely it can also make BL enzyme which needs AMP + sulbactam to treat

28
Q

HIGH LEVEL AMINOGLYCOSIDE RESISTANCE

A

Serious Enterococcal infections, are treated with Ampicillin + high- level aminoglycoside like gentamycin or streptomycin
-synergistic bactericidal effect when used together
-Amp weakens cell wall so aminogly can get in (amp can’t kill organism & gent or strep can’t get in on its own)
-some Enterococci acquire plasmid-mediated aminoglycoside destroying enzyme & develop R to high- level aminoglycoside.
*HLAR test used to detect this R & determines if synergy will be achieved with Amp + high level gent or strep.

29
Q

HLAR TEST
what is it

A

BHI agar media where half of the plate has 500ug/ml of gentamicinin it & the other half has 2000ug/ml of streptomycinin it
-0.5 McF of Enterococcus spotted on each side
-spot the Enterococcus onto a plain BHI as a growth control.
-if no growth in streptomycin let it grow for another 24 hours
-if growth in gentamin then you cant use gent or amp

QC organisms on both plates:
*An Enterococcus that is R to both aminoglycosides
*An Enterococcus that is S to both aminoglycosides
*Plates incubated in O2 35

30
Q

D TEST

A

. S aureus, BHS & S. pneumonia can be R to:
*Erythromycin (macrolide)
*clindamycin (lincosamide)
*Streptogramin B (streptogramin)

31
Q

What are some resistance methods for
S aureus, BHS & S. pneumonia

A

Acquire Erm gene: codes for production of methylase enzyme (production can be constitutive or inducible). Enzyme makes organism R to all 3 antibiotics R = MLSb resistance.

macrolide and streptogramin
Acquire msrA gene: increases efflux of antibiotic out of cell. Makes org R to macrolide & streptogramin but doesn’t affect clindamycin referred to as M-resistance.

32
Q

NDUCIBLE CLINDAMYCIN RESISTANCE

A

Detects organisms with erm gene that show their R to clindamycin in vivo only after are induced to produce methylase enzyme.

*Erythromycin is a strong inducer of methylase production.

*In vitro AST results with inducible strains would show erythromycin R but clindamycin S.

*But treatment of patient with clindamycin would fail because it is actually R.

*Test clindamycin in the presence of an inducer by doing a D test.

33
Q

WHEN TO DO A D TEST

A

Way to detect inducible R to clindamycin in an organism that has the erm gene

*Grow organism in presence of an inducer which is erythromycin

1.A BHS, S. pneumoniae or a Staphylococcus tests as erythromycin R and clindamycin S or I by routine AST testing

2.If the patient is penicillin allergic (because erythromycin or clindamycin would be used to treat these individuals)

3.If patient is already being treated with clindamycin or erythromycin

34
Q

D TEST PROCEDURE FOR INDUCIBLE CLINDAMYCIN R

A

-0.5 McF and streak for lawn of growth
-15- g erythromycin disk μin proximity to a 2- g μclindamycin disk on the agar plate.
-Media and disc placement varies based on organism
*Plate incubated 20-24hrs at 35oC, CO2 for Strep & O2 for Staph

*Look for flattening of zone (D formation) in area between Erythromycin & Clindamycin disks = inducible Clindamycin R.

35
Q

NTERPRETATION OF D TEST RESULTS KNOW FOR A TEST - RECOGNIZE THE RESULTS
D test neg

A

no flattening
zoi but not touching oO

M type resistance due to msrA gene
Only R to E due to efflux mechanism S to CL

36
Q

NTERPRETATION OF D TEST RESULTS FOR A TEST - RECOGNIZE THE RESULTS
Constitutive MLSb resistance

A

no flattening
R to both E & CL due to an erm gene expressed all the time(codes for methylase)

37
Q

NTERPRETATION OF D TEST RESULTS
FOR A TEST - RECOGNIZE THE RESULTS
D test pos

A

Flattening
D
Inducible MLSb type resistance- due to erm gene (codes for methylase) R to both E & CL –but CL R only seen in the presence of E inducer

38
Q

STREPTOCOCCUS PNEUMONIAE AST

A

*AHS, bile soluble & OPT S (zone ≥14mm)

*If AHS is bile soluble, set up KB D2, don’t wait for your OPT result D

AST testing for Penicillin:
*Penicillin susceptibility determined by oxacillin disc KB -performed on MHA w sheep blood incubated at 350 C in CO2.
*Can’t use Pen disc because doesn’t reliably detect all resistant strains.
*If Ox zone is <20mm = possible Pen R
*Must confirm by doing a Penicillin MIC before reporting it as Pen R.
*Use PEN E test or M.I.C.E

39
Q

What is an MIC how is it done

A

*The lowest concentration (ug/ml) of antibiotic that prevents visible growth of an organism.

*Prepare solutions of antibiotic at increasing concentrations & then add a constant amount of bacteria.
*First tube with no visible growth is the MIC

40
Q

What is the relationship between the MIC and the zone diameters in KB

A

*MIC and zone of inhibition are inversely correlated
*Susceptible organisms= have low MICs but large zones of inhibition
*Resistant organisms= have high MICs but small or no zone of inhibition

If organism has a high MIC it means it took a more concentrated amount of antibiotic to prevent its growth so is resistant. Will have no zone or a small zone of inhibition with KB.

If organism has a low MIC it means even very low concentrations of antibiotic were able to prevent growth and so it is susceptible.
Will have a large zone of inhibition with KB

Micro240 (7 decks)

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