AST

Question 1

Define AST

Answer 1

Antimicrobial susceptibility testing is a lab procedure designed to assess the effectiveness of specific antimicrobial agents

Question 2

What are the three main purposes of AST

Answer 2

Guiding Clinical Therapy

Antibiotic Resistance Awareness

Identify and Monitor Resistance Profiles

Question 3

Talk about the role of AST in guiding clinical therapy

Answer 3

Guides appropriate antimicrobial therapy based on the identiffied pathogens and their resistance patterns i.e. what they are susceptible too

Means targeted therapy rather than relying on broad-spectrum antimicrobials

Improve treatment outcomes, reduce side effects and shorten recovery times

Oftten comes too late to actually impact treatment decisions i.e. by the time we have the results the critical phase of illness has already passed

Question 4

Talk about the role of AST in awareness for antibiotic resistance

Answer 4

AST provides information about the resistance profiles of bacteria within a specific population or geographical area

This knowledge is essential for clinicians to understand the local resistance trends and make informed decisions

e.g. penicillin resistant S. pneumonia where there can be 50% resistance in some regions but only about 5-10% in Ireland thus penicillin is the recommended treatment option in Ireland

Question 5

Talk about the role of AST in identifying and monitoring resistance profiles

Answer 5

Detect pathogens that exhibit resistance to specific antimicrobials, including multi-drug-resistant organisms (MDROs)

Preventing the spread of resistant infections in hospitals and the broader community (Infection Control) - become increasingly more difficult (1.2 days on average in hospital)

Reveal trends in resistance patterns, showing which antimicrobials are becoming less effective over time

Monitor the emergence of resistant strains, adjust treatmnet guidelines and develop new policies for infection control

Question 6

What are the four different ways AST can be carried out?

Answer 6

Phenotypic AST

Genotypic AST

Mechanistic AST

Expert Rules

Question 7

What is phenotypic ASt?

Answer 7

Measurement of the minimum inhibitory concentration (MIC) and the interpretation of breakpoints

EUCAST breakpoints

Question 8

What is genotypic AST

Answer 8

Detection of specific resistance genes such as mecA, vanA/B, Big 5 CPE that confer resistace

Question 9

What is mechanistic AST

Answer 9

Detection of resistance mechanism such as Beta-lactamase detection

Can infer success of antibiotics from this

Question 10

What are expert rules?

Answer 10

Conditional rules that link specific resistance mechanisms to treatment decisions

e.g. rules that guide the VITEK

Involves looking at a resistance profile and making assumptions from this

Pros and cons to this as the landscape of resistace is constantly changing hence the need to mannually check VITEK sens

Question 11

What are the two different ways phenotypic AST can be carried out?

Answer 11

Qualitative:
- Disc diffusion

Quantitative:
- Macro broth
- Micro broth
- Micro well e.g. VITEK automated microwells or microtitres for fungi
- Etest

Question 12

Talk about the disc diffusion method of AST, principle and development

Answer 12

The diameter of the inhibition zone is inversely proportional to the minimum inhibitory concentration (MIC) i.e. the larger the zone the less resistant

Based on the standardised Kirby-Bauer disc diffusion approach developed in 1966 and adapted for EUCAST mIC

Question 13

What does MIC stand for?

Answer 13

minimum inhibitory concentration

Question 14

Talk about how you would carry out disc diffusion AST

Answer 14

Identify colony of interest -> since AST is species specific its important to ID correctly

Select agar: Mueler Hinton Agar for non-fastidious, MG + 5% defibrinated horse blood + 20 mg/L B-NAD for fastidious

Ensure agar is batch accepted

Ensure agar is at room temperature and dry

Inoculate with a heavy 0.5 McFarland standard

Maintain 15 minute rule

Apply disks within 15 mins

Incubate within 15 mins

Question 15

What agar is used for fastidous disc diffusion

Answer 15

mueller hinton agar + 5% mechanically defibrinated horse blood + 20 mg/L B-NAD

Question 16

Talk about batch acceptance of MH agar, give two examples of where there can be issues with agar

Answer 16

Each new batch must be tested with ATCC strains to ensure they fall within EUCAST QC ranges

Variations in Ca2+ and Mg2+ can affect aminoglycoside inhibition zones for P. aeruginosa

Excess thymine/thymidine can reduce trimethoprim-sulfamethoxazole zones for E. faecalis

Question 17

Talk about drying and storage of agar plates for disc diffusion

Answer 17

Agar must be at room temperature prior to inoculation

Surfaces must be dry i.e. not straight from fridge

Excess moisture can cause fuzzy zone edges and/or haze

Question 18

How should Disc Diffusion agar be inoculated?

Answer 18

A heavy inoculum of 0.5 McFarland should be made of -> corresponds to 102 x10^8 CFU/ml of an E. coli

According to EUCAST you should select well -isolated colonies from overnight growth on non-selective medium

4-6 hour incubation is also suitable

Question 19

Why should you not pick from selective agar?

Answer 19

Selective agar contains antimicrobials already - hene the need for a purity plate prior to AST

Question 20

What is the 15-15-15 rule

Answer 20

Inoculate agar within 15 mins of making suspension

Apply discs within 15 mins of inoculating

Incubate agar within 15 mins of applying discs

Question 21

How does disc diffusion for gram negs compare to that of gram positives?

Answer 21

For gram-negative bacteria, remove excess fluid by pressing and turning the swab against the inside of the tube to avoid over-inoculation

For gram-positive theres no need to do this

Question 22

What is the only exception to the 0.5 MacFarland for disc diffusion?

Answer 22

S. pneumoniae is suspended to a 0.5 McFarland from blood agar but to 1.0 if from a chocolate agar plate

(google this)

Question 23

How should discs by applied for disc diffusion?

Answer 23

Apply disks within 15 minutes of inoculation

Disks must be in close and even contact with the agar surface

The number of disks on a plate should be limited to avoid overlapping of zones and interference between agents

It is important that zone diameters can be reliably measured

5 to 6 disks max per plate

Question 24

How should you incubate disk diffusion plates?

Answer 24

Invert plates making sure disks stay attached to surface

Incubate at 35 degrees in air with 4-6% CO2 for 18+_2 hours

AST must be read within 18 hours of incubation hence why E. Colis were put up after lunch and read first thing in the morning

Question 25

How should you read disk diffusion

Answer 25

First check for confluent lawn, dont want any gaps in lan

For MH agar hold plate over a black, non-reflective surface and read from the back with reflected light

For MH-F agar read from the front with the lid removed illuminated with reflected light

Measure diameter of zone of inhibition to the nearest mm

Question 26

What should you do if you have distinct colonies within your zone?

Answer 26

Check for purity -> is it the same organism -> repeat if not

If cultures are pure then take colonies into account when reading result

Should subculture any colonies within the zone and repeat AST

Question 27

How should you read proteus AST

Answer 27

Ignore swarming of colonies

Question 28

Howshould you read haemolysis on disk diffusion

Answer 28

Read the inhibition of growth and not the inhibition of haemolysis

B-haemolysins diffuse in agar -> they are usually free from growth

a-haemolysins do not diffuse -> there is often growth within areas of a-haemolysis

Zone edges accompanied with a-haemolysis is most common with S. pneumoniae and B-lactam antibiotics

Question 29

How are zone sizes interpretted?

Answer 29

Check ATCC controls - zone diameters of ATCC control organisms must fall within accepted range

Criteria for test organism
- zone diamters of test organisms compared to interpretive clinical breakpoints for relevant species

Categorised as S, I or R

Question 30

What are some methods of zone reading?

Answer 30

Callipers
Ruler
Stencils
Semi-auromated systems
- reduces operator variability in reading plates
- provides auomatic interpretation of zone diameters
- zone readers linked to LIS

Question 31

What is the MIC, how are they used

Answer 31

The lowest concentration of an antimicrobial agent that prevents visible growth of a microorganism after a specified period

Clinical breakpoints determine if organism is suseptible, susceptible at increased exposure or resistant

Question 32

What is meant by susceptible?

Answer 32

Organism is susceptible at standard dosing regimen

i.e. there is a high likelihood of therapeutic success using a a standard dosing regimen of the agent

Question 33

What is susceptible, increased eposure?

Answer 33

There is a high likelihood of therapeutic success because exposure to the agent is increased by adjusting the dosing regimen or by itc concentration at the site of infection

It can be used but must increase conentration -> prevents heavy reliance on newer drugs in an attempt to reduce resistance against these

Question 34

What is defined as resistant

Answer 34

A miroorganism is categorised as Resistant when there is a high likelihood of therapeutic failure even when there is increased exposure

Question 35

What does a EUCAST NA result mean?

Answer 35

It means EUCAST wont stand over any result that falls within these breakpoints

Its usually seen with drugs used for screenining moreso than treatment e.g. cefoxitin

Question 36

What is an area of technical uncertainty (ATU)?

Answer 36

A term used by EUCAST to indicate potential confusion in interpreting antimicrobial susceptibiliy testing (AST) results, whether through MIC determination or disk diffusion

It is defined by specific MIC values or inhibition zone diameters -> it is not a susceptibility category like susceptible (S) etc

Question 37

What should you do with an area of technical uncertainty in the lab?

Answer 37

It should be a caution to the lab staff that the result falls within a range where interpretation is challenging due to the positioning of breakpoints which can lead to inconsistent result

Importantly the ATU is not due to testing errorss

There is no required action if you get one of these results but if you suspect an error then It is recommended that you repeat the method or try another method

Results should be reported as “uncertain” with a comment

Question 38

What are expert rules?

Answer 38

Conditional rules that link specific resistance mechanissm to treatment decisions

e.g. mecA in methicillin resistant staph aureus suggests resistance to multiple beta-lactam antibiotics

These rules are constantly changing ang being updated

classes organisms as either expected resistant phenotype (intrinsic resistance) or expected susceptible phenotype

Question 39

Give an example of how expert rules change over time

Answer 39

ESBLS were historically resistant against cefotaxime but now it depends on MIC

Question 40

Talk about expected resistant phenotype (Intrinsic Resistance) using expert rules

Answer 40

Refers to species where the vast majority >90% are resistant to specific antibiotics regardless of the isolate’s origin e.g. Bacteroides ID

If a laboratory result shows susceptibility it should be viewed with skepticismm and testing is generally discouraged

Typically results for these antibiotics are reported as resistant without actual testing as resistance is considered intrinsic - clinicians should be advised not to use such antibiotics fot these species

Question 41

Talk about expected susceptible phenotype (Intrinsic Resistance) using expert rules

Answer 41

Describes species that are almost always (>99%) susceptible to a given antibiotic, as resistance mechanisms of clinical relevance have not been documented

A resistant test result should raise suspicion and prompt a review of the species ID or susceptibility testing method

If a resistant result is believed to be due to an acquired resistance mechanism, it should be confirmed through reference methods, and ideally, genome sequencing

Question 42

How is AST Quality Controlled?

Answer 42

Control tests should be set up and checked daily, or at least four times per weak, for antibiotics which are part of routine panels

Control tests should always be read and evaluated before reporting ressults for clinical isolates

Each day that tests are set up, examine the results of the last 20 consecutive tests

Examine results for trends and for zones falling consistently above or below the target

If two or more of 20 tests are out of range investigation is required

Question 43

What is quantitative AST

Answer 43

AST based on MIC - refers to the minimum concentration of an antimicrobial agent required to inhibit the grorwth of a microorganism

Before automated systems such as Vitek, MICs were only done for serious infections, slow-growing organisms or fastidious organisms

Question 44

How do you establish an MIC, give some exaples of how theyre done

Answer 44

MICs are established by exposing the test organism to a range of twofold dilutions of the antimicrobial agent in an appropriate culture system:
- Macro-broth
- Micro-broth dilution
- Gradient diffusion method: Etest
- Automated-micro-well-dilution-Vitek (2 fold gradiant increase)

Question 45

What is the reference method for MIC determination?

Answer 45

Agar Dilution MIC is the gold standard

Question 46

How is agar dilution MIC carried out?

Answer 46

Antimicrobial agents are incorporated into molten agar plates at twofold serial dilutions

Bacteria are inoculated as spots on the surface of he agar

Endpoint interpretation:
- MIC is defined as the lowest concentration of the antimicrobial at which no visible growth is observed
- individual colonies or a fine haze of growth should be disregarded

Nobody actually does this - way too tedious and time consuming -> have to either incorporate antimicorbials in the agar or through spot inoculum

Question 47

Talk about broth dilution method

Answer 47

Incorporate a range of twofold serial dilutions of the anitmicrobial into the broth

For macrobroth dilution (now obselete) use over 1 ml of broth in tubes

For microbroth dilution only 50-100ul of broth is used in mirotitre plates

Question 48

Talk about Etests

Answer 48

Test isolate is exposed to a continuous antimicrobial gradient on an inert plastic strip that diffuses into the agar

After incubation an elliptical zone of inhibition forms at the MIC

Follows EUCAST guidelines for disc diffusion

Relies on diffusion yielding optimal results with organisms that grow well overnight

Expensive though at 3euro per strip, control isolates must be tested separately

Question 49

Talk about Automated AST

Answer 49

VITEK is based on micro-well dilution methods

Carries out both ID and AST

Results in 4-16 hours

Mainly hands-off

Has to be adapted for Eucast guidelines as it will follow american guidelines - hence the need for a scientist to check results

Vitek 2XL in most labs or the phoenix in beaumont