antibiotic sensitivity testing skills in microbiology in
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Antibiotic sensitivity testing , Skills in MicrobiologyTRANSCRIPT
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Dr.T.V.Rao MD 1
ANTIBIOTIC SENSITIVITY TESTINGSKILL BASED LEARNING
Dr.T.V.Rao MD
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Dr.T.V.Rao MD 2
Uses 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.
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Dr.T.V.Rao MD 3
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.
What is the goal of Antibiotic Sensitivity
testing?
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Dr.T.V.Rao MD 4
Components 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.
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Dr.T.V.Rao MD 5
Why 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
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Dr.T.V.Rao MD 6
Introduction 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)
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Dr.T.V.Rao MD 7
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
Introduction
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Dr.T.V.Rao MD 8
Which organisms to test? What methods to use? What antibiotics to test? How to report results?
What Does the Laboratory Need to Knowabout Antimicrobial Susceptibility Testing
(AST) ?
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Dr.T.V.Rao MD 9
Routine Susceptibility
Tests Disk diffusion
(Kirby Bauer) Broth micro-
dilution MIC NCCLS
reference method
Etest
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Dr.T.V.Rao MD 10
Preparing 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.
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Dr.T.V.Rao MD 11
Choosing 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
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Dr.T.V.Rao MD 12
Table 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
EnterobacteriaceaeUrinary
SulfonamideTrimethoprimCo-trimoxazoleAmpicillinNitrofurantoinNalidixic acidTetracycline
NorfloxacinChloramphenicolGentamicin
Blood and tissues AmpicillinChloramphenicolCotrimoxazoleTetracyclineGentamicin
CefuroximeCeftriaxoneCiprofloxacinPiperacillinAmikacin
Pseudomonas aeruginosa PiperacillinGentamicinTobramycin
Amikacin
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Dr.T.V.Rao MD 13
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
Antimicrobial Susceptibility Testing
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Susceptibility Testing Methods
InoculateMH plate
Place diskson agar plate
Incubate plate18-24 hr, 35 CMeasure and record zone of inhibition around each disk
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Dr.T.V.Rao MD 15
Diffusion 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
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Dr.T.V.Rao MD 16
Disc 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
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Dr.T.V.Rao MD 17
Examining purity of plateSelect the Colonies from Pure
Isolates
Reflected light
Transmitted light
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Dr.T.V.Rao MD 18
Disk Diffusion
Test
Select colonies
Prepare inoculumsuspension
Prepare inoculumsuspension
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Dr.T.V.Rao MD 19
Prepare the Material
for Inoculation
Standardize inoculumSuspension as per Mac farland standard
Mix well
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Dr.T.V.Rao MD 20
Swab the plate with
optimal sample
Remove sample
Swab plate
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Dr.T.V.Rao MD 21
Select the Disks and
Apply
Select disks
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Dr.T.V.Rao MD 22
Incubate
Overnight
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Dr.T.V.Rao MD 23
Disc 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
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Dr.T.V.Rao MD 24
Read the Results
with Precision
TransmittedLight
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Dr.T.V.Rao MD 25
Disc 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
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Dr.T.V.Rao MD 26
Look 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.
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Dr.T.V.Rao MD 27
Disc Diffusion MethodReporting 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
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Dr.T.V.Rao MD 28
Factors Affecting Size of Zone of Inhibition
Inoculum density
Timing of disc application
Temperature of incubation
Incubation time
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 < 35 oC
Ideal 16-18 hours; less time does not give reliable results
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Dr.T.V.Rao MD 29
Factors 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
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Dr.T.V.Rao MD 30
Factors Affecting Size of Zone of Inhibition
Potency of antibiotic discs
Composition of medium
Acidic pH of medium
Alkaline pH of medium
Reading of zones
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
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Dr.T.V.Rao MD 31
Quality 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
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Dr.T.V.Rao MD 32
Quality 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 )
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Dr.T.V.Rao MD 33
Quality 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
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Dr.T.V.Rao MD 34
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
Need for Modified Methods
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Dilution 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
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Antimicrobial susceptibilitytesting using micro-broth
dilutions
•
•
••••
•• •
•
•
••
96 well microtiter plate
ug/ml64 32 16 8 4 2
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Broth 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
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Dr.T.V.Rao MD 38
Creating Dilutions
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Broth Dilution Method
Day 1
Add 1 ml of test bacteria (1*106 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.= 5*105 CFU/ml
Incubate 35 oC, o/n
128 64 32 16 8 4 2 C1 C2
64 32 16 8 4 2 1 C1 C2
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Broth Dilution Method
Day 2Record visual turbidity
Subculture non-turbid tubes to agar plates (use 0.01 ml standard loop)
MIC = 16 mg/l
64 32 16 8 4 2 1 C1 C2
0.01 ml (spread plate), Incubate 35 oC, o/n
64 32 16
Day 3Determine CFU on plates:At 16 mg/ = 700 CFU/ml > 0.1% of 5*105 CFU/ml
MBC = 32 mg/l
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Broth Dilution Method
100% of original bacterial conc. = 5*105 CFU/ml
0.1% = [(5*105)*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 500*0.01 = 5 CFU
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Dr.T.V.Rao MD 42
Broth 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
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Micro 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
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Agar 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
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Agar Dilution MethodProcedure
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
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Dr.T.V.Rao MD 46
Minimal inhibitory
concentration
The lowest concentration of antimicrobial agent that inhibits the growth of a bacterium
Interpret: Susceptible Intermediate Resistant
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Dr.T.V.Rao MD 47
Clinical Conditions when
MICs are Useful Endocarditis Meningitis Septicemia Osteomyelitis Immunosuppressed patients (HIV,
cancer, etc.) Prosthetic devices Patients not responding despite “S”
Reports
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Dr.T.V.Rao MD 48
Inoculum Preparation
MIC Testing (NCCLS Reference Method)
Standardize inoculum suspension
Final inoculum concentration3 – 5 x 105
CFU/ml(3 – 5 x 104
CFU/well)
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Dr.T.V.Rao MD 49
Select Micro titration
plate and prepare optimal inoculum
Micro dilution MIC tray
Prepare inoculumsuspension
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Dr.T.V.Rao MD 50
Dilute & mix inoculum
suspension
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Dr.T.V.Rao MD 51
Pour inoculum into reservoir
and inoculate MIC tray
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Dr.T.V.Rao MD 52
Incubate overnight
Do not forget to check the purity of Inoculum
Inoculate purity plate
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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
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- +
643216 8 4 2 1
>64
0.5
Read MICs
>64
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Dr.T.V.Rao MD 55
The 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.
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Dr.T.V.Rao MD 56
E 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.
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Antimicrobial Gradient
TestingE-test®
Read platesafter
recommendedIncubation
Read MICwhere elipse
intersectsscale
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Dr.T.V.Rao MD 58
MIC of the Bacteria can be
read Directly
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MIC on a stripabbiodisk.com
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5-Jan-06 Chiang Mai University 60
Serum Susceptibility Tests
To determine drug concentration in the patient’s serum = MIC*SIT The Serum Inhibitory Titer (SIT)
The highest dilution of patient’s serum that inhibit bacteria
To determine the ability of drug in the patient’s serum to kill bacteria The Serum Bactericidal Level (SBL)
The lowest dilution of patient’s serum that kills bacteria Technically Demanding
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Antibiotic Sensitivity
testing can be done with automation
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VITEK 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.
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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
What is the Role of Microbiology Departments
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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.
References
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For Articles of Interest on Antibiotics follow me on
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Created by Dr.T.V.Rao MD for ‘e’
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