prof.dr.ihsan alsaimary -college of medicine - university of basrah -iraq ihsanalsaimary@gmail.com 009647801410838

1. Laboratory Aspects of
Antimicrobial Chemotherapy
Patrick Kimmitt

2. Aims of Presentation
• Answers to the following questions:-
• (1) Why do we test antimicrobial
susceptibility?
• (2) How do we perform antimicrobial
susceptibility tests?
• (3) How can we detect resistance
mechanisms?
• (4) Why & how do we assay antimicrobial
serum levels?

3. Deciding on whether to use an
antibiotic:
• Day 1 Clinical assessment
• Day 2 Positive microbiology
• Day 3 Antibiotic sensitivity tests
• Day 1: guess the disease, guess the bug, guess
the sensitivity pattern
• Day 2: guess the sensitivity pattern
• Day 3: know the full picture: BUT TOO LATE?

4. Antibiotics and the Laboratory
• If there was no antibiotic resistance, there
would be no need for a microbiology lab!
• Infections could be treated “syndromically”

5. Why do we test antimicrobial
susceptibility?
• To direct & predict antimicrobial
chemotherapy.
• To review & monitor epidemiological
trends.
• To set national & local antibiotic policies.
• To test the activity of a new antimicrobial
agent.
• To presumptively identify isolates.

6. But remember…
• Other factors are very important when we
choose an antibiotic
• Will it get to where the infection is?
• Bioavailability
• Cost
• Toxicity
• Likelihood of development of resistance
• Etc…

7. How do we perform antimicrobial
susceptibility tests?
• We can use a number of methods including:-
• Disc susceptibility tests - Kirby-Bauer
- Stokes’
- BSAC.
• Agar Breakpoint method.
• Minimum Inhibitory Concentration (MIC) – Tube
MIC or E-tests.
• Automated methods – Vitek.
• Molecular methods – PCR.

8. Disc Susceptibility Tests
• Agar surface evenly inoculated with the test
organism.
• Antibiotic filter paper discs applied to the plate.
• Plates are incubated & antibiotics diffuse into the
agar.
• Antibiotic concentration decreases at increasing
distance from disc.
• Circular zone of growth appears.
• Size of zone of inhibition indicates susceptibility
of organism.

9. Disc dispenser & inoculated plate

10. Stokes’ Comparative Method
• Developed in the U.K (1972).
• A variety of media can be used including
Iso-sensitest agar (ISA), ISA & 5% horse
blood & Chocolate ISA.
• Based on dense not confluent growth.
• Use suspension of organism in broth
equivalent in density to an overnight broth
culture.
• Inoculate fastidious organisms direct.

11. Stokes’ Comparative Method
• Use NCTC (National Collection of Type
Cultures) controls e.g. NCTC 6571 Staph
aureus, NCTC 10602 Ps. aeruginosa,
NCTC 10418 E.coli.
• Using a rotary plater apply the control
suspension on the outer edge & the test
suspension in the centre, leaving a gap for
the discs.

12. Stokes’ method
Control
organism Test
organism
Antibiotic
discs

13. Stokes’ Comparative Method

14. Stokes’ Comparative Method
• Interpretation based on comparison between
zones seen with the test organism & those of the
known sensitive control.
• Sensitive = zone radius of test, larger, equal, or
not more than 3mm smaller than the control.
• Intermediate = zone radius more than 3mm, but
smaller than the control by more than 3mm.
• Resistant = zone radius of 3mm or less.

15. Disadvantages of Stokes’ Method
• Interpretation not valid for β lactamase-
producing staphylococci (research for
practical) or for tests with polymyxin,
augmentin, teicoplanin or ciprofloxacin.
• No correlation of zone diameter with the
MIC of the organism.
• No standard method for inoculum
preparation- a heavy inoculum decreases
zone of inhibition.

16. Disadvantages of Stokes’ Method
• No standard method for media or
incubation conditions- pre-incubation
decreases the zone of inhibition, pre-
diffusion increases the zone of inhibition.
• Unreliable for detection of resistance to
new antibiotics or newer resistance
mechanisms (ESBL’s).
• No consistent method between labs,
therefore no consistent epidemiology data.

17. BSAC Method
• Developed in the U.K in 1998 by BSAC
working party, through statistical
regression analysis of zone diameter &
MIC data, on hundreds of strains.
• A full up-to-date version of the method is
available at www.bsac.org.uk.
• Use ISA and/or ISA & 5% horse blood &
20mg/l NAD.
• Use standard 0.5 McFarland inoculum

18. BSAC Method
• Use this diluted inoculum to seed the media
(using a rotary plater or by streaking in 3
directions).
• Use standard inoculation & incubation criteria.
• Use standard antibiotic quality controlled discs.
• Use published BSAC tables to interpret zone
sizes.
• http://www.bsac.org.uk/_db/_documents/version
_7_1_february_2008.pdf

19. Examples of BSAC Method

20. BSAC Method
• Interpretation is based on semi-confluent growth
of the organism.
• Zones sizes can be measured using a template /
ruler / electronic callipers / automated zone
reader with a scanner & camera (Aura Image,
Oxoid).
• The method is subject to weekly NCTC control
checks for each panel of antibiotics tested.
• These controls are checked against published
values.

21. Rationale of BSAC method
• Based on the
relationship between
the zone diameter of
the disc diffusion test
and the MIC.
• The clinical
breakpoint can be set
as equivalent to a
stated zone diameter
(mm)

22. Advantages of the BSAC Method
• BSAC working party continually review &
update the data.
• Standardised method of reporting.
• Increases the accuracy of epidemiological
data.
• Attempting to standardise protocols and
breakpoints throughout Europe (EUCAST)
– April 2008

23. Minimum Inhibitory Concentration
(MIC)
• The MIC is the lowest concentration of the
antimicrobial required to inhibit growth of the
organism.
• It is used to determine the quantitative activity of
an antimicrobial.
• It is used to confirm resistance or equivocal
results.
• It is used in cases of prolonged treatment or
endocarditis to adjust the dose of therapy.
• It is used to determine the susceptibility of slow-
growing organisms e.g anaerobes

24. Tube MIC
• Set up a series of antibiotic doubling
dilutions in tubes containing liquid media
(ISA or ISA with lysed blood).
• For example 128mg/l, 64, 32, 16, 8, 4, 2,
1, 0.5, 0.25, 0.125, 0.
• Set up tubes for test organism & NCTC
control organism.

25. Tube MIC
• Add standard organism inoculum to each
tube.
• Include an antibiotic free tube i.e organism
only.
• Include an organism free tube i.e antibiotic
only.
• Incubate tubes overnight.
• Examine for presence of growth by
shaking each tube & observing turbidity.

26. Tube MIC
• Check antibiotic free tube has growth.
• Check organism free tube has no growth.
• Check the NCTC control gives the
recommended MIC.
• The MIC is the first tube dilution without
visible growth.
• The tube MIC is very labour intensive,
difficult to get right & prone to error.

27. E-tests (AB Biodisk(Sweden))
• A commercial alternative to tube MIC.
• Consists of a plastic strip 6cm by 0.5cm in
size.
• Exponential gradient of antimicrobial dried
on one side.
• MIC scale printed on the other side.
• The range corresponds to fifteen 2-fold
dilutions.

28. E-tests
• Follow manufacturers’ instructions for
inoculum preparation, media
recommendations & incubation conditions.
• MIC interpretation made where growth of
inhibition ellipses the strip.
• Most E-test require examination with a
hand-lens to look for minute colonies
intersecting the strip.

29. Examples of E-tests

30. Examples of E-tests

31. Automated Methods
• Three main methods are in use in the U.K.
• These include the Vitek (Biomerieux), the
Phoenix (Becton-Dickinson) & the
Mastascan Elite (Mast).
• This presentation will focus on the Vitek,
please research the other two methods.

32. Vitek II
• The Vitek I was originally designed by
NASA for use as an on-board space
exploration test system.
• It is based on the use of small thin plastic
cards each containing many wells linked
by capillaries.
• These cards are available as susceptibility
& identification cards.

33. Vitek cards

34. Vitek modules
• The Vitek II consists of:-
• a robotic filling module whereby a standard
suspension of the organism in saline is drawn up
via a vacuum into the card.
• an incubator / reader module containing a
carousel to hold the cards & a photometer to
measure optical density of the sensitivity cards &
the biochemical colour changes of the
identification cards

35. Vitek modules
• A computer module analyses the growth curve &
generates an algorithm devised MIC value. It
also analyses the biochemical id & compares it
to a database.
• An “expert” software analysis module
recognises new / unusual / inconsistent results,
highlights “alert” organisms (e.g. MRSA, VRE,
Gentamicin resistance).
• The “expert” system has built-in antibiotic
interpretation rules.

36. Vitek modules

37. Vitek advantages / disadvantages
• Can give rapid “expert” results for
identification & susceptibility.
• It can be directly linked to the LIMS.
• It decreases the incidence of operator
error & can be operated by an MLA.
• It is very expensive, approx £7 for an id &
sens.
• It needs regular software updates & expert
analysis by a BMS.

38. Why & how do we assay
antimicrobial serum levels?
• We assay to ensure adequate therapeutic levels
& to avoid the accumulation of toxic levels.
• Most antimicrobial agents have a large
therapeutic index & are given in large doses
without causing harm.
• The aminoglycosides, the glycopeptides & some
antifungals have a narrow therapeutic range
which can be close to the toxic range.

39. Dosing interval and steady state plasma concentration
Time
Drugconcentrationinplasma
Therapeutic
range
Therapeutic
failure
Therapeutic
failure

40. Antibiotic Assays
• These agents can damage the 8th cranial
nerve = ototoxicity = deafness.
• They can also damage the kidneys =
nephrotoxicity = renal failure.
• Serum samples are often taken pre-dose
to determine toxicity & post-dose to
determine therapeutic activity.
• We can assay antibiotics in 3 main ways –
bioassay, immunoassay and HPLC

41. Immunoassays
• There are several commercial
immunoassay methods available.
• Most are based on competitive binding of
antibody for antigen (serum) & labelled
antigen (kit).
• The most common commercial method is
the TDX.

42. TDX Analyser

43. Calibration curve
• A calibration curve of polarisation versus
concentration is set up within the TDX
using known concentration calibrators.
• Internal controls are run with each assay
to ensure the curve is correct.
• The assay level is calculated by the TDX
via extrapolation from the calibration
curve.

44. Summary
Antibiotic
Assays
Detecting
resistance
mechanisms
Molecular
methods
Automated
methods
MIC tests
Breakpoint
testing
Disc testing
Lab Aspects
Of Antimicrobial
Chemotherapy

45. References
• British Society for Antimicrobial Chemotherapy
www.bsac.org.uk
• Medical Bacteriology: A Practical Approach Ed P.Hawkey
& D.Lewis, Oxford University Press, 2003.
• Antibiotics & Chemotherapy: Anti-Infective Agents & their
use in therapy R.Finch et al Churchill Livingstone, 2002.
• Textbook of Diagnostic Microbiology C.R Mahon &
G.Manuselis Saunders & Co Ltd 2000.
• Antimicrobial Chemotherapy D. Greenwood Oxford
Medical Publications 1999.
• Clinical Microbiology; E. J. Stokes, G.L. Ridgway &
M.W.D Wren: Arnold; 1993