Guide to understand

Antibiotic susceptibility
testing (AST)

Dr. Neelima Kulshrestha

Clinical microbiologist
Shekhar hospital
Role of clinical microbiologist…
• Culture of sample
• Identification of relevant pathogen
• Antibiotic sensitivity testing
• Guide and advise treating team.
TIMELINE OF ANTIBIOTICS
Fosfomycin

Minocycline

NDM

KPC

β lactamases
(TEM1 and TEM2)

S.S. Tang et al. Advanced Drug Delivery Reviews 2014

3
Reviving old antibiotics. J Antimicrob Chemother 2015
“RESISTANT BUGS, NO DRUGS.”
 Why AST it required ?
• An useful guide to use antibiotics correctly.
• Antibiotics are very precious →Judicious use of
antibiotics is essential.
• Resistance - ↑morbidity & mortality d/t
treatment failures and ↑ health care costs.
 Principle of AST?
• Antimicrobial contained in a reservoir disc is
allowed to diffuse out into medium and
interact in culture plate which is freshly
seeded with the test organisms.
What is Antibiotic susceptibility testing ?
Sample (Day 1)

Culture over appropriate plating media

Kept in incubator for certain hours

Growth over plating media (Day 2)

Put AST over appropriate media and incubate

Get the result of AST (Day 3)

 Factors to consider while performing a
susceptibility test

• Body site
• Quality of specimen
• Host’s immune status
 Body Site
• Not routinely performed for commensals.
Eg.
E. coli in stool sample
E. coli from a blood culture
Viridans gp streptococci in throat swab
Throat swabs Streptococcus pyogenes (group
A Streptococcus)
CONS from skin surface swab
 Quality of the specimen
• Isolation of organism in pure culture-less likely
to be contamination.eg.
Urine sample- Presence of ˃ 2spp. at ˃ 10⁵
CFU /ml suggest contamination.
Pure culture of E. coli at ˃ 10⁵ CFU /ml-tested.
 Host’s immune status
• In an immunocompromised patient, normal
microbiota may require testing
eg.
• CONS from blood culture of paediatric patient.
• Patient allergic to penicillin with group A
Streptococcus infection testing for Erythromycin
is warranted.
 How to select antibiotics in AST?
• First
Antimicrobial agents included in formulary and
prescribed by physicians routinely.
• Second
Acc/t CLSI depending on:-
 organism group
 site of infection (e.g., CSF, blood, urine or faeces).
 Intrinsic resistance (e.g., vancomycin in Gram-
negative bacilli)
 Guidelines we follow in INDIA
• CLSI-Clinical laboratory standard institute.
• Develop clinical and laboratory practices and
promote their use worldwide.
Updated annually
 CLSI grouping of Abs for AST:-
• Group A Primary Test and Report
• Group B Primary Testing but report selectively as
 Resistant to grp A
 Selected specimen source eg. 3rd gen
cephalosporins for enteric bacilli from CSF
 Polymicrobial infection
 Allergic
 Failure to respond
 CLSI categorization…
• Group C Supplemental Report Selectively as
Strains resistant to several primary drugs
Treatment of unusual organism
(extraintestinal Salmonella spp.)
• Group U urine sample
 Methods available
Diffusion
• Stokes method
• Kirby-Bauer method
Dilution (MIC)
• Broth dilution
• Agar Dilution
Diffusion & Dilution
• E-Test method
Diffusion method
 Diffusion method
• Antibiotics are added to plate after an
organism is streaked onto agar.
• Drug diffuses from paper discs into agar.
• After a period of incubation, a zone of
inhibition can be seen.
• Disc diffusion method- Kirby-Bauer method
most practical method.
Diffusion method -Kirby-Bauer method
 Disadvantage of Kirby-Bauer method
• Cannot tell exact concentration of antibiotic
needed to achieve a therapeutic result.
Dilution (MIC) method
 Indications
• To confirm AST results of disc diffusion tests.
• For testing antimicrobial sensitivities of -
slow growing bacteria such as TB bacilli
bacteria for which diffusion test is not
standardized
• Accurate therapeutic dose determination as in
treatment of bacterial endocarditis.
 Dilution (MIC) method
• Common dilution tests include broth dilution
and agar dilution.
• Involves challenging the organism to
antimicrobial agents in a liquid environment.
• Bacteria are placed onto tubes containing a
two fold dilution of antibiotic.
 Agar Dilution
32 16 8 4 2 1
mcg/ml mcg/ml mcg/ml mcg/ml mcg/ml mcg/ml
 Agar Dilution (contd.)
• A quantified concentration of microorganism
is on an agar plate.
• MIC is measured in this example, where the
antibiotic has a MIC of 32 mcg/mL.
 Broth dilution (contd.)
• 96 wells are filled with 0.1 mL of twofold
dilution conc. of antimicrobial agent in broth.
• A quantified amount of microorganism is
added to each well, with varying conc. of 8
antibiotics (A–H).
• Black circles→ no bacterial growth.
• Growth may be seen as turbidity, a haze, or a
pellet in the bottom of the well.
• MIC-A is 32 mcg/mL and for antibiotic C, 0.5
mcg/mL.
 Antimicrobial
Agent

Sterile Control

Growth Control

0.25
0.50
1
2
4
8
16
Broth dilution

32
64
128
Broth microdilution 96 well plate
Tray-reading stand with magnifying
mirror (left) and
light box (right).
 Broth dilution (contd.)
• Disadvantages-
labor-intensive
time-consuming
potential procedural error
Diffusion & Dilution
(E-Test method)
 E test (Epsilometer test)
• Manual in vitro diagnostic device.
• To determine MIC.
Principle
• Strip with a predefined gradient of
antibiotic is applied to agar plate.
 E test (contd.)
• Instantaneous release of
antimicrobial from strip into agar.
• Form a stable and continuous
gradient in vicinity of strip.
• MIC value is read from the scale
where the ellipse edge intersects the
strip.
Interpretation of E-test
 Reporting of AST ?
Sensitive -
organism is inhibited by the serum conc. of drug
that is achieved using the usual dosage.

Resistant-
organisms are resistant to usually achievable
serum drug.
 Reporting…

Intermediate-
• Organisms are inhibited only by the maximum
recommended dosage.
 Case 1.
• A 50 yrs old diabetic patient admitted to ICU
with lower limb cellulitis with septicemic
shock. Blood culture was sent.
• Report of blood culture shows Staphylococcus
aureus resistant to Cefoxitin.
• What does it mean?
• What will be the line of treatment ?
Staph. aureus resistant to Cefoxitin means?
• Resistant to the following drugs-
 β-lactam/ β-lactamase inhibitor combination
(amoxicillin-clavulanate,pip-tazobactam etc.)
Oral cephalosporins
Parenteral cephalosporins
Carbapenems.
Line of treatment?
 Line of treatment for MRSA-
1. Vancomycin
2. Teicoplanin
3. Linezolid
4. Quinupristin/dalfopristin
 Case 2.
• A 65 yrs old patient was admitted to our hospital with fever
and respiratory distress. The patient was intubated and kept
on ventilator. Three days after admission his condition
worsened.
• Tracheal aspirate showed heavy growth of Acinetobacter
baumannii.
• Sensitive to amoxicillin-clavulanate, ertapenem, fosfomycin,
meropenem, colistin.
• Resistant to Ampicillin-sulbactum, TMP-SMZ.

• Is this AST report reliable ?

 Intrinsic resistance
• Acinetobacter baumannii is intrinsically
resistant to amoxicillin-clavulanate,
ertapenem, fosfomycin.
• Is the innate ability of a type of bacteria
species to resist the action of an antibiotic as a
consequence of bacteria's structural or
functional characteristics.
 Case 3.
• A 80 yrs old patient admitted to our ICU with
septicemic shock. Blood culture was sent and
antibiotics were started. Report of blood
culture shows growth of E. coli sensitive to
several drugs.
• We did MIC and…..
AST report
 How are MICs…(contd.)
• For example: A strain of Escherichia coli has an MIC
of 2 μg/mL for amoxicillin and for cefovecin.
• Looking at the dilutions for amoxicillin, at 2 μg/mL,
this strain of E. coli is four dilutions away from the
breakpoint.
• For cefovecin, the same strain of E. coli at an MIC of
2 μg/mL is two dilutions away from the breakpoint.
• So, based on MICs, this strain of E. coli is more
susceptible to amoxicillin than cefovecin.
 How are MICs used ?
• Lowest concentration of an antibiotic that
inhibits the growth of bacteria.
• The choice of antibiotic should be based on
the MIC number,
the site of infection,
how far the MIC is from the breakpoint
 Automated methods for AST
• Rapid (5 to 15 hours) or overnight (16 to 24
hours) susceptibility test results.
• Provide-
 rapid and accurate quantitative result of AST
 provide MIC
 Types of automated methods
• There are 4 automated instruments presently
cleared by the FDA.
1. MicroScan WalkAway- broth disc dilution

2. BD Phoenix Automated system

3. The Vitek 2 System Broth
4. Sensititre ARIS 2X microdilution
 Principles
• First-Based on turbidimetric detection of
bacterial growth using a photometer.
• Second-Detection of hydrolysis of a
fluorogenic growth substrate incorporated in a
special test medium.
The Vitek 2 System cassette
 MALDI-TOF
• Matrix-assisted laser
desorption ionization–time-of-
flight mass spectrometry.
• Based on detection of mass of
molecules.
• USE
Rapid identification of
bacteria and fungi.
Principle
 Near-future alternatives for AST
1. Calorimetrics
2. Cantilever technology
3. FACS ( fluorescence-activated cell sorting)
4. Magnetic bead spin
5. MALDI-TOF
6. Microdroplets
7. Next-generation sequencing
 Take home messages:-
• AMR is one of the most-significant health care
problems of the new millennium.
• Ideally, cultures should be drawn prior to the patient
receiving antibiotics esp. in ICU.
• Clinical microbiologist plays a central role in
optimizing the therapeutic management of patients.
• Kirby-Bauer-most practical and useful method for
AST.
• To get rapid result automated methods are gaining
popularity.
• The CLSI zone size and MIC interpretive criteria are
established by analysis of 3 kinds of data:
• (1.) microbiologic data, including a comparison of MICs
and zone sizes on a large number of bacterial strains,
including those with known mechanisms of resistance that
have been defined either phenotypically or genotypically;
• (2) pharmacokinetic and pharmacodynamic data;
• (3) clinical studiesstudies results (including comparisons of
MIC and zone diameter with microbiological eradication
and clinical efficacy) obtained during studies prior to FDA
approval and marketing of an antibiotic.
 Epidemiologic
cut off values (ECVs)
• Are critical MICs other than the MICs used to judge a
bacterial susceptibility.
• It is several MIC dilutions lower than the MIC used to
judge a bacterium susceptible to an antimicrobial.
• Triggers a concern that bacteria is developing
resistance to the antimicrobial.
• Indicates-
 discontinuing the use of drug
 Use of the drug for specific patients only
• In vitro test conditions can have an influence on in vivo
antimicrobial activity
• sub-MIC effects
• postantibiotic effects,
• protein binding,
• effects on organism virulence
• variations in redox potential at sites of infection,
• and the pharmacokinetic changes resulting from different
• drug levels in blood and at the site of infection over time
• Breakpoints (or interpretative criteria) are the
values that determine the categories of
susceptible, intermediate and resistant.
• Breakpoint is the highest plasma conc. of drug
that can safely be achieved in patient.
• Other factors to take into consideration are
the site of the infection,
frequency and route of administration,
 and cost of the antibiotic.
• An antibiotic breakpoint is the dilution where
bacteria begin to show resistance.