Title: Antibiotic Sensitivity Testing
1ANTIBIOTIC SENSITIVITY TESTINGSKILL BASED
LEARNING
2Uses of Antibiotic Sensitivity Testing
- Antibiotic sensitivity test A laboratory test
which determines how effective antibiotic therapy
is against a bacterial infections. - Antibiotic sensitivity testing will control the
use of Antibiotics in clinical practice - Testing will assist the clinicians in the choice
of drugs for the treatment of infections.
3What is the goal of Antibiotic Sensitivity
testing?
- The goal of antimicrobial susceptibility testing
is to predict the in vivo success or failure of
antibiotic therapy. Tests are performed in vitro,
and measure the growth response of an isolated
organism to a particular drug or drugs. The tests
are performed under standardized conditions so
that the results are reproducible. The test
results should be used to guide antibiotic
choice. The results of antimicrobial
susceptibility testing should be combined with
clinical information and experience when
selecting the most appropriate antibiotic for our
patients.
4Components of Antibiotic Sensitivity Testing
- 1.The identification of relevant pathogens in
exudates and body fluids collected from patients - 2. Sensitivity tests done to determine the degree
of sensitivity or resistance of pathogens
isolated from patient to an appropriate range of
antimicrobial drugs - 3. Assay of the concentration of an administered
drug in the blood or body fluid of patient
required to control the schedule of dosage. -
5Why Need Continues for Testing Antibiotic
Sensitivity
- Bacteria have the ability to develop resistance
following repeated or subclinical (insufficient)
doses, so more advanced antibiotics and synthetic
antimicrobials are continually required to
overcome them. - Antibiotic sensitivity testing is essential part
of Medical Care
6Introduction
- Susceptibility test, main purposes
- As a guide for treatment
- Sensitivity of a given m.o. to known conc. of
drugs - Its concentration in body fluids or tissues
- As an epidemiological tool
- The emergence of resistant strains of major
pathogens (e. g. Shigella, Salmonella typhi) - Continued surveillance of the susceptibility
pattern of the prevalent strains (e. g.
Staphylococci, Gram-negative bacilli)
7Introduction
- Methods for antimicrobial susceptibility testing
- Indirect method
- cultured plate from pure culture
- Direct method
- Pathological specimen
- e.g. urine, a positive blood culture, or a swab
of pus
8What Does the Laboratory Need to Knowabout
Antimicrobial Susceptibility Testing (AST) ?
- Which organisms to test?
- What methods to use?
- What antibiotics to test?
- How to report results?
9Routine Susceptibility Tests
- Disk diffusion (Kirby Bauer)
- Broth micro-dilution MIC
- NCCLS reference method
- Etest
10Preparing for Testing
- Inoculum preparation
- - Number of test organisms can be determined
using different methods - Direct count (Microscopic examination)
- The optical density (OD) at 600 nm
(Spectrophotometry) - Plate count making dilution first
- Turbidity standard (McFarland) routinely
performed.
11Choosing the Appropriate Antibiotic
- Drugs for routine susceptibility tests
- Set 1 the drugs that are available in most
hospitals and for which routine testing should be
carried out for every strain - Set 2 the drugs that are tested only
- at the special request of the physician
- or when the causative organism is resistant to
the first-choice drugs - or when other reasons (allergy to a drug, or its
unavailability) make further testing justified
12Table 1 Basic sets of drugs for routine
susceptibility tests (http//w3.whosea.org/)
Set 1 Set 2
Staphylococcus Benzyl penicillinOxacillinErythromycinTetracyclineChloramphenicol GentamicinAmikacinCo-trimoxazoleClindamycin
Intestinal AmpicillinChloramphenicolCo-trimoxazoleNalidixic acidTetracycline Norfloxacin
Enterobacteriaceae Urinary SulfonamideTrimethoprimCo-trimoxazoleAmpicillinNitrofurantoinNalidixic acidTetracycline NorfloxacinChloramphenicolGentamicin
Blood and tissues AmpicillinChloramphenicolCotrimoxazoleTetracyclineGentamicin CefuroximeCeftriaxoneCiprofloxacinPiperacillinAmikacin
Pseudomonas aeruginosa PiperacillinGentamicinTobramycin Amikacin
13Antimicrobial Susceptibility Testing
- Diffusion method
- Put a filter disc, or a porous cup/a bottomless
cylinder containing measured quantity of drugs on
the a solid medium that has been seeded with test
bacteria - Dilution method
- vary amount of antimicrobial substances
incorporated into liquid or solid media - followed by inoculation of test bacteria
14Susceptibility Testing Methods
Incubate plate 18-24 hr, 35 C Measure and record
zone of inhibition around each disk
Inoculate MH plate
Place disks on agar plate
15Diffusion Method
- Disc diffusion method The Kirby-Bauer test
- Antibiotic-impregnated filter disc
- Susceptibility test against more than one
antibiotics by measuring size of inhibition zone
- 1949 Bondi and colleagues? paper disks
- 1966 Kirby, Bauer, Sherris, and Tuck ? filter
paper disks - Demonstrated that the qualitative results of
filter disk diffusion assay correlated well with
quantitative results from MIC tests
16Disc Diffusion Method
- Procedure (Modified Kirby-Bauer method National
Committee for Clinical Laboratory Standards.
NCCLS) - Prepare approximately. 108 CFU/ml bacterial
inoculum in a saline or tryptic soy broth tube
(TSB) or Mueller-Hinton broth (5 ml) - Pick 3-5 isolated colonies from plate
- Adjust the turbidity to the same as the McFarland
No. 0.5 standard. - Streak the swab on the surface of the
Mueller-Hinton agar (3 times in 3 quadrants) - Leave 5-10 min to dry the surface of agar
17Examining purity of plateSelect the Colonies
from Pure Isolates
Transmitted light
Reflected light
18Disk Diffusion Test
Prepare inoculum suspension
Prepare inoculum suspension
Select colonies
19Prepare the Material for Inoculation
Standardize inoculum Suspension as per Mac
farland standard
Mix well
20Swab the plate with optimal sample
Remove sample
Swab plate
21Select the Disks and Apply
Select disks
22Incubate Overnight
23Disc Diffusion Method
- Place the appropriate drug-impregnated disc on
the surface of the inoculated agar plate - Invert the plates and incubate them at 35 oC, o/n
(18-24 h) - Measure the diameters of inhibition zone in mm
24Read the Results with Precision
Transmitted Light
25Disc Diffusion Method
- Measurement of the diameters of inhibition zone
- Measure from the edge where the growth stats, BUT
there are three exceptions - With sulfonamides and co-trimoxazole, ignore
slight growth within the zone - Certain Proteus spp. may swarm into the area of
inhibition - When beta-lactamase producing Streptococci are
tested, zone of inhibition are produced with a
heaped-up, clearly defined edge, regardless of
the size of the inhibition zone, they should be
reported as resistant
26Look at the Charts for establishing the zones of
Sensitivity
- The zone sizes are looked up on a standardized
chart to give a result of sensitive, resistant,
or intermediate. Many charts have a corresponding
column that also gives the MIC (minimal
inhibitory concentration) for that drug.
27Disc Diffusion Method Reporting the Results
- Interpretation of results
- By comparing with the diameters with standard
tables - Susceptible
- Intermediate susceptible
- Low toxic antibiotics Moderate susceptible
- High toxic antibiotics buffer zone btw resistant
and susceptible - Resistant
28Factors Affecting Size of Zone of Inhibition
- Larger zones with light inoculum and vice versa
- If after application of disc, the plate is kept
for longer time at room temperature, small zones
may form - Larger zones are seen with temperatures lt 35 oC
- Ideal 16-18 hours less time does not give
reliable results
- Inoculum density
- Timing of disc application
- Temperature of incubation
- Incubation time
29Factors Affecting Size of Zone of Inhibition
- Size of the plate
- Depth of the agar medium (4 mm)
- Proper spacing of the discs (2.5 cm)
- Smaller plates accommodate less number of discs
- Thin media yield excessively large inhibition
zones and vice versa - Avoids overlapping of zones
30Factors Affecting Size of Zone of Inhibition
- Deterioration in contents leads to reduced size
- Affects rate of growth, diffusion of antibiotics
and activity of antibiotics - Tetracycline, novobiocin, methicillin zones are
larger - Aminoglycosides, erythromycin zones are larger
- Subjective errors in determining the clear edge
- Potency of antibiotic discs
- Composition of medium
- Acidic pH of medium
- Alkaline pH of medium
- Reading of zones
31Quality Assurance in Antibiotic Susceptibility
Testing
- Visit - WHO-Regional Office for South East Asia
website - Medium Mueller-Hinton agar plates
- Enterococcus faecalis (ATCC 29212 or 33l86) and a
disc of co-trimoxazole? 20 mm in diameter of the
inhibition zone - Procedure Modified Kirby-Bauer method
recommended by National Committee on Clinical
Laboratory Services (NCCLS) - Susceptibility test with quality control strains
32Quality Assurance in Antibiotic Susceptibility
Testing with Control strains
- Susceptibility test with quality control strains
- for every new batch of Mueller-Hinton agar
- Staphylococcus aureus (ATCC 25923)
- Escherichia coli (ATCC 25922)
- Pseudomonas aeruginosa (ATCC 2785 )
33Quality Assurance in Antibiotic Susceptibility
Test
- Salient features of quality control
- Use antibiotic discs of 6 mm diameter
- Use correct content of antimicrobial agent per
disc - Store supply of antimicrobial discs at -20 oC
- Use Mueller-Hinton medium for antibiotic
sensitivity determination - Use appropriate control cultures
- Use standard methodology for the test
34Need for Modified Methods
- Modified Methods in Disc diffusion for
Antibiotic sensitivity testing to be used for
detections of following bacterial isolates - 1 MRSA
- 2 ESBL
- 3 Enterobacteriaceae and Gram negative bacteria
and Carbapenems resistant using Modified Hodge
test
35Dilution Method
- Minimum Inhibition Concentration (MIC)
- The lowest concentration of antimicrobial agent
that inhibits bacterial growth/ multiplication - Minimum Bactericidal Concentration (MBC) or
Minimum Lethal Concentration (MLC) - The lowest concentration of antimicrobial agent
that allows less than 0.1 of the original
inoculum to survive
36Antimicrobial susceptibilitytesting using
micro-broth dilutions
ug/ml 64 32 16 8 4 2
96 well microtiter plate
37Broth Dilution Method
- Procedure
Making dilutions (2-fold) of
antibiotic in broth Mueller-Hinton, Tryptic Soy
Broth - Inoculation of bacterial inoculum, incubation,
overnight - Controls no inoculum, no antibiotic
- Turbidity visualization ? MIC
- Sub culturing of non-turbid tubes, overnight
- Growth (bacterial count) ? MBC
38Creating Dilutions
39Broth Dilution Method
Day 1 Add 1 ml of test bacteria (1106 CFU/ml)
to tubes containing 1 ml broth and concentration
of antibiotic (mg/l)
Controls C1 No antibiotic, check viability on
agar plates immediately C2 No test bacteria
Bacterial conc. 5105 CFU/ml Incubate 35 oC, o/n
40Broth Dilution Method
Day 2 Record visual turbidity Subculture
non-turbid tubes to agar plates (use 0.01 ml
standard loop) MIC 16 mg/l
0.01 ml (spread plate), Incubate 35 oC, o/n
Day 3 Determine CFU on plates At 16 mg/ 700
CFU/ml gt 0.1 of 5105 CFU/ml MBC 32 mg/l
41Broth Dilution Method
- 100 of original bacterial conc.
- 5105 CFU/ml
- 0.1
- (5105)0.1/100 CFU/ml
- 500 CFU/ml
- The bacteria count should be less than 5 CFU on
agar plate subcultured with 0.01 ml - 5000.01 5 CFU
-
42Broth Dilution Method are Technically Difficult
- Disadvantages
- Only one antibiotic one organism can be tested
each time - Time-consuming
- Solutions??
- Agar dilution method
- Disc diffusion method
- Micro broth dilution method
43Micro broth Dilution Method
- Micro dilution plates
- Micro dilution/ Micro broth dilutions
- 96 wells/ plate simultaneously performed with
many tests organisms/ specimens, less reagent
required - Manually prepared
- Commercially prepared
- Frozen or Dried/ lyophilized
- Consistent performance but high cost
- May suffer from degradation of antibiotic during
shipping and storage
44Agar Dilution Method
- Procedure
- Making dilutions of antimicrobial agent in melted
media and pouring plates - One concentration of antibiotic/ plate
- Possible for several different strains/plate
64 uGu/ml 32 ug/ml 16 ug/ml
45Agar Dilution Method
- Procedure
- Inoculation of bacterial inoculum (McFarland No.
0.5) - Using a replicating inoculator device called A
Steers-Foltz replicator - Delivers 0.001 ml of bacterial inoculum
- Incubation
- Spot of growth
MIC
32 ug/ml
46Minimal inhibitory concentration
- The lowest concentration of antimicrobial agent
that inhibits the growth of a bacterium - Interpret
- Susceptible
- Intermediate
- Resistant
47Clinical Conditions when MICs are Useful
- Endocarditis
- Meningitis
- Septicemia
- Osteomyelitis
- Immunosuppressed patients (HIV, cancer, etc.)
- Prosthetic devices
- Patients not responding despite S Reports
48Inoculum Preparation MIC Testing (NCCLS
Reference Method)
- Standardize inoculum suspension
- Final inoculum concentration
- 3 5 x 105 CFU/ml
- (3 5 x 104 CFU/well)
49Select Micro titration plate and prepare optimal
inoculum
Prepare inoculum suspension
Micro dilution MIC tray
50Dilute mix inoculumsuspension
51Pour inoculum into reservoir and inoculate MIC
tray
52Incubate overnightDo not forget to check the
purity of Inoculum
Inoculate purity plate
53 Optimal Use of Purity Plates
- Sub final test suspension to non-selective medium
(after inoculating MIC test) - Streak for isolation (avoid several specimens per
plate - may not reveal contaminants if no
isolated colonies) - Examine before reading MIC (usually at 16-20 h)
- Re-incubate if Antibiograms questionable
54Read MICs
-
0.5
1
2
4
8
16
32
64
gt64
gt64
55The gradient technique, Etest
- Etest is a well established AST method in
microbiology laboratories around the world. The
Etest technique comprises a predefined gradient
of antibiotic concentrations on a plastic strip,
and can be used to determine the Minimum
Inhibitory Concentration (MIC) of antibiotics,
antifungal agents and antimycobacterial agents.
56E test MIC Reports are helpful in Critical
management decisions
- Quantitative MIC data is a prerequisite for the
management of critical infections, including
sepsis, especially among critical care patients.
Etest is particularly valuable in such
situations, when on-scale MICs are needed for
treatment decisions.
57Antimicrobial Gradient TestingE-test
Read plates after recommended Incubation
Read MIC where elipse intersects scale
58MIC of the Bacteria can be read Directly
59MIC on a strip abbiodisk.com
60Serum Susceptibility Tests
- To determine drug concentration in the patients
serum MICSIT - The Serum Inhibitory Titer (SIT)
- The highest dilution of patients serum that
inhibit bacteria - To determine the ability of drug in the patients
serum to kill bacteria - The Serum Bactericidal Level (SBL)
- The lowest dilution of patients serum that kills
bacteria - Technically Demanding
61Antibiotic Sensitivity testing can be done with
automation
62VITEK 2 Automates Reporting of Resistance
- Integrated in the VITEK 2 system is the Advanced
Expert System (AES), a software which validates
and interprets susceptibility test results, and
detects antibiotic resistance mechanisms. The AES
Expert System is the most developed software
system in this field, and is capable of
identifying even emerging and low-level
resistance.
63What is the Role of Microbiology Departments
- Each laboratory should have a staff member with
the time, interest, and expertise to provide
leadership in antibiotic testing and resistance.
This person would read relevant publications,
network with other laboratories, and evaluate
potentially useful tests to detect new forms of
resistance before new CLSI-recommended tests
become available - - Ken Thomson, Emerging Infect. Dis., 2001
64References
- 1Usanee Anukool (Ph.D.) Clinical
Microbiology,AMS, - Chiang Mai University
- 2National Committee For Clinical Laboratory
Standards. 1998. NCCLS document M100 - S8 .
Performance Standards for Antimicrobial
Susceptibility Testing. 8th edition, NCCLS,
Waynae, Pa.
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