Antibiotic susceptibility testing Flashcards
What are some examples of enzymatic cleavage/inactivation forms of bacterial resistance?
- Beta lactams and aminoglycoside antibiotics
2. Beta lactamases and aminoglycoside modifying enzymes
What are some examples of bacterial resistance due to altered receptors/binding proteins?
- altered penicillin binding proteins
- Strep pneumo resistance to Penicillin
- ** MRSA resistance to Methicillin**
What are some examples of bacterial resistance due to altered permeability?
Pseudomonas resistance to aminoglycosides
(Gram neg bacilli alter influx and efflux pumps stopping passage through porins)
What is an example of bacterial resistance due to a metabolic block?
Enterococcus resistance to TMP/SXT
What organization sets the standards for antibiotic susc testing?
CLSI: Clincial laboratory standards institute
- publish new standards yearly
- decide which are appropriate abx to test
- Set QC standards and proper procedures
What are necessary steps for preparation of an organism for testing?
- Organism must be in LOG phase
- Must use a pure culture (1 organism)
- Amount of organism is set to 0.5 MacFarland Standard (=turbidity of 10^8 bacteria CFU/mL)/ or can use spectrophotometer
- Incubate at 35C for 16-24hrs
What are some cons to Kirby Bauer testing?
-Is qualitative NOT quantitative (reports whether sensitive or resistant, does NOT give MIC)
-Is manual and susceptible to human error (interpretation size and set up)
-If ions too high–> Aminoglycosides are falsely resistant;
If ions too low–> Aminoglycosides are falsely susc
How do you measure a double zone in Kirby Bauer testing?
Measure the inside or more resistant zone.
How do you arrive at MIC for Kirby Bauer testing?
Must use regression analysis to calculate MIC value related to KB zone size
What are pros/cons of E-tests?
Pros: -Flexible, can use any strip -Quantitative, gives MIC outright -Can use for anaerobes, yeast and bacteria Cons: -Higher cost, $3 per strip -Still have that damn human error!
How is broth dilution performed?
Organism further diluted to 5x the 0.5 MacFarland standard (10^5 org)
- inoculated into microtiter trays with growth medium and known 2 fold dilutions of antibiotic.
- *Must have 4 or more dilutions to be true MIC?
What is the difference between MIC and MBC?
MIC: minimum INHIBITORY concentration= minimal amount of antibiotic needed to stop growth or an organism, doesn’t mean it’s not still around
MBC: minimum bactericidal concentration= minimum amount of abx to KILL organism
How is MIC vs MBC determined?
MIC: dilution at which org is no longer growing in well
MBC: plate every dilution at and after to MIC to see if microorganism grows on the plate; no growth= MBC
What is tolerance?
When an organism is inhibited by antibiotics but cannot be killed, fortells resistance. MBC/MIC ratio will be <32
How are bacteria tested for beta-lactamase?
With a filter paper disk that has a chromogenic cephalosporin (Nitrocefin).
Interpretation: Red color= breakdown in beta lactam ring POSITIVE for beta-lactamase
Negative= retains yellow color
What antibiotics are bacteria resistant to if the beta lactamase test is positive?
Ampicillin, Penicillin, Cephalosporin
Which are two key organisms that may produce beta lactamase? What abx does a positive test imply resistance to?
- Haemophilus influenzae–> 20 -40% produce beta lactamase, if so resistant to Ampicillin
- Bacteriodes fragilis group–> primary mech of resistance is beta lactamase production, resistant to Penicillin
How is methicillin resistance tested for S. aureus?
- Staph. aureus is tested against OXACILLIN because it is more stable than methicillin and methicillin is no longer commercially available.
* * If resistant to Ox then IS RESISTANT TO ALL BETA LACTAMS AND ALL CEPHALOSPORINS ARE REPORTED AS RESISTANT**
What is the mech of resistance for MRSA**??
mecA gene encodes penicillin binding proteins
What are two screening methods used for MRSA?
1) 6mcg/ml of Oxacillin and 4% NaCl (enhances detection of resistance) plated to Mueller Hinton agar
2) Cefoxitin screens (Cefoxitin is a better inducer of the mecA gene and therefore more sensitive at detecting resistance by production of PBPs)
What is the theory behind the D test?
MRSA that is susceptible to Clindamycin has the ability to attain inducible resistance if certain enzymes are present. The D-test uses Erythromycin resistance to determine if the MRSA has occult resistance to Clindamycin
How do you interpret a D test?
A blunted edge between the Erythromycin and Clindamycin test means Clinda resistance can be induced by erythromycin.
Enterococci are naturally resistant to???
- Cephalosporins
- Clindamycin
- Tmp/SXT
What two drugs have a synergistic effect against enterococci?
Ampicillin/ Gentamycin (beta lactam/aminoglycoside)
How do you screen for synergy? Why do it?
put organism in a tube with 500 mcg/ml of gentamycin, if no growth then it is susceptible
**If growth present is not amenable to Gentamycin and Gent can cause nephrotoxicity and ototoxicity
How do enterococci acquire resistance to Vancomycin?
It is plasmid mediated
- vanA: Enterococcus faecium
- vanB: Enterococcus faecalis
How do you test for VRE?
can use Kirby Bauer, E test or automated systems
Drugs of choice-Linezolid and Synercid*
Which organisms are capable of producing extended spectrum beta-lactamases? How is the resistance acquired?
E. coli, Klebsiella and Proteus
-plasmid mediated (Tem 1 beta lactamase plasmid)
What is the significance of testing positive for extended spectrum beta-lactamase production?
You must report all CEPHALOSPORINS and PENICILLINS as RESISTANT
-typically still susceptible to carbapenems (Imipenem), Cephamycins (Cefotetan) and beta lacam inhibitors (Tazobactam or Clavulinic acid)
What is the significance of Klebsiella pneumonia Carbapenemase (KPC)?
Not only resistant to Cephalosporins or Penicillins, but also Carbapenems.
- Test with Ertapenem, Imipenem, Carbapenem
What is two step testing for Strep pneumo?
- Test S. pneumo for Penicillin susceptibility against Oxacillin (more sensitive), if susceptible, then susc to Penicillin; IF RESISTANT
- Confirm with MIC test against Penicillin (E-test, broth dilution, etc)
What is the significance of the MIC values when testing S. pneumo for Penicillin susc?
Different breakpoints for type of infection:
CSF: Resistant if >= 2mcg/ml
Resp/blood: Resistant if >= 8 mcg/ml
What drugs can you use if S. pneumo is resistant to Penicillin
Cefotaxime, Vancomycin or a quinolone
What strain of C. difficile is hypervirulent?
NAP1 strain; more relapse, more serious infection, community acquired
What toxins are responsible for disease in C. difficile?
Toxin A=enterotoxin (fluid)
Toxin B= cytotoxin
Test of choice previously EIA, now PCR***
When amplifying in PCR, what does it mean when your negative control is positive? What must you do?
Means there is contamination in the system, you must rerun the ALL patients from the patients ORIGINAL sample
Why should each patient sample have an internal control in addition to the positive and negative control used for the assay?
The internal control will suggest issues with the sample and not the system if negative such as inhibitors to amplification (Vagisil, zinc)
When is the term “indifferent” used as a result in PCR amplification?
When you cannot tell if the results are positive or negative (I’m assuming this is after re-running the sample?)
