1. AST(other notes from class)

How does the D-zone test work?

On an agar plate covered in staph/strep isolate, place a erythromycin disk near a clindamycin disk

Pos result = D-shaped zone of inhibition around clindamycin disk = the erythromycin has induced clindamycin resistance

What does the D-zone test, test for?

Inducible clindomycin resistance

What are beta-lactam agents?

A category of antibiotics with beta-lactam rings (includes all the -illins, cephalosporins, carbapenams)

(methicillin, oxacillin, amoxicillin, ampicillin, carbenicillin, cefoxitin, ceftaroline, etc)

What is B-lactamase?

An enzyme that bacteria can form in response to B-lactam antibiotics (bacterial resistance, ew)

What is PBP2a?

Its a penicillin-binding protein encoded by the mecA/C gene

What does PBP2a do?

When bacteria alter their PBP gene, they can prevent binding of B-lactam antibiotics

So if a bacteria gets a plasmid that codes for PBP-2a, they become resistant to all B-lactam antibiotics (MRSA does this)

Can you screen for oxacillin-MRSA w/ agar?

Yes, with mueller-hinton screening agar containing oxacillin and NaCl

Only resistant strains should grow

Can you screen for VRSA with agar?

Yes, with vancomycin-containing agar

Only resistant strains should grow

CFU stands for?

colony forming unit

1750 CFUs, cultured w/ 0.10mL loupe, what is the concentration in CFU/mL?

1750CFU/0.01mL=

175,000CFU/mL=

1.75 x 10^5 CFU/mL

What 2 plates are used to culture urine?

Sheep blood agar and Maconkey

272 CFU cultured w/ 0.001mL loupe, concentration?

272CFU/0.001mL =

272,000CFU/mL =

2.72x10^5CFU/mL

MIC is qualitative or quantitative?

quantitative

Disk diffusion testing is qualitative or quantitative?

Qualitative

MIC stands for

minimum inhibitory concentration

AST stands for

antimicrobial susceptibility testing

AST methods

Broth macrodltn

Broth microdltn

Agar dltn

Kirby bauer testing

MBC testing

Serum bactericidal test

Vitek

E-tests

D-zone test

B-lactamase test

Oxacillin/vancomycin plates

MH agar broth/agar

Disk diffusion testing is aka

KIRBY BAUER TESTING

MBC stands for

minimum bactericidal concentration

Once MIC is determined, how is the organism interpreted?

Susceptible

Intermediate

Resistant

What are dilution methods in AST used for?

to determine the MIC

On what type of bacteria are AST tests performed?

-Isolated from clinical sample

-Determined to be cause of disease

-The susceptibility to antimicrobials is uncertain

Do we do AST if we know what antimicrobial agent is used to treat a particular infectious organism?

NO, we already know, we dont do extra testing for funsies

Name antimicrobial inactivating enzymes

B-lactamase

Extended spectrum B-lactamases

AmpC enzymes

Carbapenemases

If a bacteria is oxicillin resistant, why is it also methicillin resistant?

methilin falls under oxicillin category

How can bacteria share antibiotic resistance with each other?

Horizontal gene transfer lets bacteria share AB-resistant genes via plasmids through conjugation

What is a downside of e-tests

expense

What are e-tests useful for?

Useful for testing fastidious organisms. such as:

H. influenzae, S. pneumoniae, anaerobic bacteria

How does kirby bauer testing work? How is MIC determined by kirby bauer?

The MICs are related to the diameter of the zones of inhibition and it is the relationship between the 2 that determines the susceptibility pattern of a particular isolate

Name the 1 reference method for AST testing of anaerobes and N. gonnorrhoeae

Agar dltn test

In what clinical setting are agar dilution tests performed

research settings

Is there a limit to the antimicrobial concentrations we test?

We only look at antimicrobial concentrations that are safe to treat a patient with

Organisms that produce ESBL are able to destroy the activity of?

Ticarcillin

Carbenicillin

Piperacillin

Mezlocillin

Aztreonam

cephalosporins other than cefoxitin

Organisms that produce ESBL are usually susceptible to

Imipenem

Meropenem

Doripenem

ertapenem

cefoxitin

beta-lactamase inhibitors (clavulanic acid, tazobactam and sulbactam)

Organisms that produce ESBL are usually resistant to

Fluoroquinolones

Aminoglycosides

B-lactamase production is a means of resistance to B-lactam antibiotics for organisms such as?

Neisseria gonorrhoeae

H. influenzae

Moraxella catarrhalis

Staphylococcus spp.

Enterococcus spp.

some Bacteroides spp.

What do AmpC enzymes do?

provide resistance to:

the first, 2nd/3rd gen cephalosporins, penicillins, aztreonam

but not the 4th gen cefepime or the carbapenems

AmpC enzyme is most commonly associated w/ what bacteria

Serratia spp

Providencia spp

Citrobacter freundii

Enterobacter spp

Morganella morganii

Aeromonas spp

Pseudomonas aeruginosa (SPACE) and indole-positive Proteus spp. (SPICE)

How to interpret Susceptible result

Infecting org should respond to antibiotic therapy using recommended dosing schedule

How to interpret Resistant result

infecting org wont repspond to antibiotic therapy using the dosing schedule normally recommended

How to interpret Intermediate result

Infecting org can be susceptible if a higher antibiotic dose is used

How to interpret Non-Susceptible (NS) result

isolate that only has interpretive-susceptible critera

When to report NS

When antimicrobial agents have MICs above or zone diameters that are below the susceptible breakpoint

How to interpret Susceptible-Dose Dependant (SDD) result

implies susceptibility of an isolate is dependent on dosing regimen (e.g. higher doses, more frequent doses, both)

Broth macrodltn test procedure

2-fold serial dltn

1-2 ml volume per tube

Add bacterial suspension

Final [ ] should be 5x10^5CFU/ml

Have positive and neg control

Incubate at 35C overnight

MIC=lowest [ ] that inhibits growth

Use mueller-hinton stock for nonfastidious bacteria!

Broth microdltn procedure

2-fold serial dltn

0.1mL of agent dltn/well

Bacterial suspension innoculated into wells simultaneously

Final [bacteria] is 1-5x10^5CFU/mL

Incubate 35C overnight

MIC determined visually w/ tray reading device

What is a pro of the broth microdltn method

(Up to 15 antimicrobial agents can be tested)

Why would we use the MBC instead of the MIC at a PTs site of infection?

Healthy PT w/ good immune system --> MIC at site of infection = bacteria gets eradicated

Immunocompromised PT --> a bactericidal [ ] at infection site is needed to cure

What is the MBC test used to determine?

the bactericidal activity of a particular antimicrobial agent

What other tests is the MBC test done with?

in conjunction w/ the broth macrodltn OR broth microdltn MIC tests

How is MBC determined?

A reduction in > 99.9% of the colonies following subculture from the MIC test at the lowest possible [ ] is equal to the MBC

What does the serum bactericidal test measure?

measures the effectiveness of penicillin in serum against bacteria that caused endocarditis

How do we do QC for antimicrobial tests?

we test reference strains of bacteria (strains that represent patient clinical isolates) that have known antimicrobial susceptibility patterns bc the CLSI has their acceptable zone diameters and MICs

Give a few examples of automated susceptibility test methods

-"Hand off" incubation/reading systems

-Systems that automatically read microdltn trays

-robotic maneuvering of trays, cuvettes, adding reagents, etc

Standard media/broth for AST

Mueller hinton

(modify for fastidious bacteria though)

When do we do D-zone testing for Staphylococcus?

When a strain of S aureus has been determined to be erythromycin resistant (but clindamycin susceptible)

The oxacillin screening plate for MRSA contains what

Mueller-Hinton agar plate supplemented with 4% NaCl and 6 ug/ml oxacillin

Drug of choice to treat staphylococcal resistane

Oxacillin (and other penicillinase-resistant penicillins)

What happens if staph has a resistance to any of the penicillinase-resistant penicillins?

resistance to all of them should be concluded

What are carbapenemases?

plasmid mediated enzymes that confer resistance to all penicillins, cephalosporins, carbapenems and aztreonam.

most common pathogen with the ability to produce carbapenemases

Klebsiella pneumoniae

(E.coli can too)

If K. pneumoniae or E. coli produce ESBLs, what is the next thing you test them for? And how?

Carbapenemases

Test w/ modified Hodge Test

AmpC screening has a inducing antibiotic and a detection antibiotic for the staph D-zone test. Name those 2 antibiotics

Inducing--Cefotoxin

Detection--ceftazadime

Most common test when testing for B-lactamases

chromogenic cephalosporin named nitrocefin

B-lactamase test--> Nitrocefin Procedure

Nitrocefin soaked into filter-paper

Filter-paper is innocualted w/ organism

Pos rxn=red/brown colour = beta-lactamase activity

What should be reported if an organism produces ESBL?

resistance to all penicillins, cephalosporins and aztreonam

2 antibiotics used to treat uncomplicated enterococci infections

ampicillin or penicillin

3 antibiotics used to treat serious enterococci infections

Ampicillin/penicillin AND aminoglycoside

What do beta-lactam antibiotics do?

bind to and inactivate enzymes required for bacterial cell wall synthesis