Micro Week 5: ID & Sensi

What organisms is the Kirby-Baur disc diffusion standardized to test?

Commonly isolated, rapidly growing (non-fastidious) bacterial pathogens such as the Enterobacteriaceae, Staphylococcus, Enterococcus, Pseudomonas aeruginosa, and Acinetobacter.

What does it mean for an organism to be rapidly growing to be tested for by the KB method?

Must grow within 24-48 hours

KB: Testing and application of zone diameter interpretations for other fastidious, slow organisms should not be done as this may lead to..

false results

What are sources of error for the KB method? (refer to page 18 of handout)

• Inoculum size - increased inoculum size leads to small zones and false resistance; decreased inoculum size leads to large zones and false sensitives

• Incubation time - Increased incubation temperature leads to failure to detect MRSA as they grow slower under elevated temperatures

• Wrong media depth - increased media depth causes smaller zones and false resistance; decreased media depth causes larger zones and false sensitives. Results from improper media preparation

• Using the CO2 incubator - high CO2 decreases surface pH and will affect aminoglycosides, erythromycin and clindamycin with less activity, tetracyclines with more activity

• Excessive delay in incubating the plate after disks are placed may cause larger zones and false sensitives

• Using old beta-lactam antibiotic discs may cause small zones and and false resistance

• Using disc cartridges stored at room temperature may cause small zones and false resistance

Kirby-Baur: How does error in inoculum size affect results?

• Increased inoculum size leads to small zones and false resistance

• Decrease inoculum size leads to large zones and false sensitives

Kirby-Baur: How does an increase in incubation temperature affect results in detecting MRSA?

Leads to failure to detect MRSA as they grow slower under elevated temperatures

Kirby-Baur: How does an increase or decreased in incubation time affect results?

• Increased incubation time causes smaller zones → false resistance

• Decreased incubation time causes larger zones → false sensitives

Kirby-Baur: How does an error in media depth affect results?

• Increased media depth causes smaller zones → false resistance

• Decreased media depth causes larger zones → false susceptibility

What is MIC?

Minimal Inhibitory Concentration

• Lowest concentration of an antimicrobial drug that prevents visible growth of a microorganism (E test)

*McFarland standard

Barium sulfate precipitate in acid solution

• Gives standard turbidity to compare susceptibility inoculum preparations

The 0.5 McFarland standard is the most common. It matches a bacterial suspension of roughly..

1.5 × 10⁸ CFU/ml

You are running an MRSA screening test with cefoxitin. What if the incubator is too hot (over 37C)?

There will be no growth, resulting in a false “no MRSA” result

When a sample is tested using Phoenix, the BD Phoenix AST Indicator is mixed with the sample. What is the purpose of the indicator?

It is a resazurin-based oxidation-reduction (redox) indicator

• It acts as a visual tracker to detect active bacterial growth within the panel’s micro-wells

• If the bacteria multiply, their ongoing cellular respiration and metabolism transfer electrons to the indicator, chemically reducing it. The liquid changes from blue to pink

Phoenix: If a well containing an antibiotic remains blue, it means..

the drug successfully stopped the bacteria from growing (the bacteria are susceptible)

Phoenix: If a well containing an antibiotic turns pink, it means..

the bacteria are surviving and replicating despite the antibiotic (the bacteria are resistant)

Minor discrepancy

A testing error where the initial test method varies slightly from the reference test method. The patient treatment is not affected

After performing a susceptibility test, you report a drug as resistant. It is later found that the drug is susceptible. This is considered..

a minor discrepancy

Major discepancy

A testing error where the initial method indicates a susceptible result whereas the reference test method indicates resistant. False susceptible results could have a major impact on patient treatment.

The vitek results report a drug as susceptible. You run a confirmation test (E-test/ Phoenix) that yields a different result. What do you do?

Report the latter result

True/ False: Susceptibility can be predicted based on an ID.

False

• e.g. If a patient were to come back to the hospital with E. coli in the same wound, do not assume the same susceptibilities as the previous test. Always perform a new test.

What Gram positive organisms are testing for susceptibilities?

S. aureus, Enterococcus, S. pneumoniae, B-hemolytic strep, (Coag, neg staph, micrococcus, certain Bacillus sp. when clinically relevant)

Cephalosporins mode of action

Beta-lactam ring that attach to and block penicillin-binding proteins (PBPs) → block cell wall synthesis → lysis

Antibiotic synergy

Two or more antimicrobials having different mechanisms of action which augment each other’s effect

What drugs fit under cephalosporins?

Cephalothin, Cefuroxime, Cefotaxime, Ceftazidime, Cefepime, Ceftaroline, Cefbioprole

S. pneumoniae testing

E-test, penicillin (oxacillin disc), 3rd Gen Cephalosporin ab range differences

Carbapenums

Imipenem, Meropenem, Ertapenem, Doripenem

How do hyper-mucoid organisms affect Vitek testing?

They usually cannot be testing through Vitek due to tendency of clogging pipets

Cefanase test

A rapid test used to detect beta-lactamase in bacteria.

• The disc is impregnated with nitrocefin, a yellow chromogenic cephalosporin

• A bacterial colony is directly applied to the disc. If the bacteria produces the beta lactamase, it will cleave the beta-lactam ring of nitrocefin to produce a red pigment

ESBL testing with antibiotic discs

The tests requires the use of both cefotaxime and ceftazidime, alone and in combination with clavulanic acid. Both are set up on the same media. A => 5 mm increase in a zone diameter for either antimicrobial agent tested in combination with clavulanic acid indicates the presence of ESBL.

D-disk antibiotics

Erythromycin and Clindomycin

MRSA

Possess the MecA gene that encodes the PBP2A protein → affinity of beta-lactams antibiotics for bacteria decreases → antibiotics do not kill MRSA

Macrolides

Erythromycin, clarithromycin, azithromycin

Susceptibilities testing identifies a bacteria as vancomycin resistant. What do you do?

Confirm the results, as vancomycin resistance is highly unlikely.

MALDI theory

Identifies bacteria by checking for proteins (not DNA/ RNA)

What happens if MALDI fails?

• Alternate methods of ID may be used = GNI, GPI, Phoenix ID

• Ensure that there is enough biomass → grow plates for long enough