Rao MD

Dr.T.V.Rao MD


 test: A laboratory Antibiotic sensitivity

Uses of Antibiotic Sensitivity Testing

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.
Dr.T.V.Rao MD 2

  The goal of antimicrobial susceptibility testing is to

What is the goal of Antibiotic Sensitivity testing?

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.
Dr.T.V.Rao MD 3

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 Dr.T.V.Rao MD dosage.


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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 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, Gramnegative bacilli)
Dr.T.V.Rao MD 6

Introduction 
 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

Dr.T.V.Rao MD


What Does the Laboratory Need to Know about Antimicrobial Susceptibility Testing (AST) ?

Which organisms to test?

What methods to use?
What antibiotics to test?

How to report results?
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Routine Susceptibility Tests
Disk diffusion (Kirby Bauer)
Broth microdilution MIC
 NCCLS reference method

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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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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 firstchoice drugs  or when other reasons (allergy to a drug, or its unavailability) make further testing justified
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Table 1: Basic sets of drugs for routine susceptibility

tests (http://w3.whosea.org/)
Set 1
Staphylococcus Benzyl penicillin Oxacillin Erythromycin Tetracycline Chloramphenicol Ampicillin Chloramphenicol Co-trimoxazole Nalidixic acid Tetracycline Sulfonamide Trimethoprim Co-trimoxazole Ampicillin Nitrofurantoin Nalidixic acid Tetracycline Ampicillin Chloramphenicol Cotrimoxazole Tetracycline Gentamicin Piperacillin Gentamicin Tobramycin

Set 2
Gentamicin Amikacin Co-trimoxazole Clindamycin



Enterobacteriaceae Urinary

Norfloxacin Chloramphenicol Gentamicin

Blood and tissues

Cefuroxime Ceftriaxone Ciprofloxacin Piperacillin Amikacin Amikacin

Pseudomonas aeruginosa

Dr.T.V.Rao MD


Antimicrobial 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
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Susceptibility Testing Methods

Inoculate MH plate

Place disks on agar plate

Incubate plate 18-24 hr, 35 C Measure and record zone of inhibition around each disk

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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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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Examining purity of plate Select the Colonies from Pure Isolates

Reflect ed light Transmitted light

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Disk Diffusion Test

Prepare inoculum suspension

Prepare inoculum Select colonies suspension

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Prepare the Material for Inoculation

Standardize inoculum Suspension as per Mac farland standard

Mix well

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Swab the plate with optimal sample 
Remove sample
Swab plate

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Select the Disks and Apply

Select disks

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Incubate Overnight

Dr.T.V.Rao MD


Disc Diffusion Method

Place the appropriate drugimpregnated 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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Read the Results with Precision 
Transmitted Light

Dr.T.V.Rao MD


Disc Diffusion Method
 Measurement of the diameters of inhibition


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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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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Disc 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

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Factors Affecting Size of Zone of Inhibition

 Inoculum density  Timing of disc application

 Larger zones with light

inoculum and vice versa plate is kept for longer time at room temperature, small zones may form

 If after application of disc, the

 Temperature of incubation  Incubation time

 Larger zones are seen with

temperatures < 35 oC

 Ideal 16-18 hours; less time
Dr.T.V.Rao MD

does not give reliable results

Factors Affecting Size of Zone of Inhibition
 Size of the plate

 Smaller plates 

accommodate less number of discs

 Depth of the agar

medium (4 mm)

 Thin media yield

excessively large inhibition zones and vice versa zones

 Proper spacing of

the discs (2.5 cm)

 Avoids overlapping of

Dr.T.V.Rao MD


Factors Affecting Size of Zone of Inhibition
 Potency of antibiotic


 Deterioration in contents leads

to reduced size

 Composition of


 Affects rate of growth,

diffusion of antibiotics and activity of antibiotics methicillin zones are larger

 Acidic pH of medium

 Tetracycline, novobiocin,  Aminoglycosides,

 Alkaline pH of


erythromycin zones are larger
determining the clear edge

 Reading of zones

 Subjective errors in

Dr.T.V.Rao MD

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

Dr.T.V.Rao MD


  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 )

Quality Assurance in Antibiotic Susceptibility Testing with Control strains

Dr.T.V.Rao MD


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
Dr.T.V.Rao MD 33

Need 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
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Dilution Method

Concentration (MIC) Minimum Inhibition
 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

Antimicrobial susceptibility testing using micro-broth dilutions 
ug/ml 64 32 16 8 4 2

• • • • • • • •
96 well microtiter plate

• •

Broth Dilution Method

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

Creating Dilutions 

Dr.T.V.Rao MD


Broth Dilution Method

128 64 32 16 8 4 2 C1 C2

Day 1 Add 1 ml of test bacteria (1*106 CFU/ml) to tubes containing 1 ml broth and concentration of antibiotic (mg/l)







1 C1 C2

Controls: C1 = No antibiotic, check viability on agar plates immediately

Bacterial conc.= 5*105 CFU/ml Incubate 35 oC, o/n

C2 = No test bacteria

Broth Dilution Method
Day 2 64 32 16 8 4 2 1 C1 C2 Record visual turbidity Subculture non-turbid tubes to agar plates (use 0.01 ml standard loop)

0.01 ml (spread plate), Incubate 35 oC, o/n

MIC = 16 mg/l Day 3 Determine CFU on plates: At 16 mg/ = 700 CFU/ml > 0.1% of 5*105 CFU/ml MBC = 32 mg/l




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


Broth Dilution Method are Technically Difficult

Disadvantages :
Only one antibiotic & one organism can be tested each time Time-consuming

 Agar dilution method  Disc diffusion method  Micro broth dilution method

Dr.T.V.Rao MD


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

Agar Dilution Method
 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


Agar Dilution Method
 Procedure

Inoculation of bacterial inoculum (McFarland No. 0.5)

Using a replicating inoculator device called “A SteersFoltz replicator” Delivers 0.001 ml of bacterial inoculum

 

Incubation Spot of growth
32 ug/ml

Minimal inhibitory concentration

The lowest concentration of antimicrobial agent that inhibits the growth of a bacterium

 Susceptible  Intermediate  Resistant
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Clinical Conditions when MICs are Useful
Meningitis Septicemia Osteomyelitis Immunosuppressed patients (HIV, cancer, etc.) Prosthetic devices Patients not responding despite “S” Reports
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Inoculum Preparation
MIC Testing
(NCCLS Reference Method)
Standardize inoculum suspension Final inoculum concentration 3 – 5 x 105 CFU/ml (3 – 5 x 104 CFU/well)
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Select Micro titration plate and prepare optimal inoculum

Micro dilution MIC tray

Prepare inoculum suspension

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Dilute & mix inoculum suspension

Dr.T.V.Rao MD


Pour inoculum into reservoir and inoculate MIC tray

Dr.T.V.Rao MD


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

Read MICs
0.5 1 2 4 8 16 32 64

- +

>6 4

>6 4

Etest is a well established

The gradient technique, Etest® 
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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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 Dr.T.V.Rao MD decisions.


Antimicrobial Gradient Testing E-test®

Read plates after recommended Incubation Read MIC where elipse intersects scale

MIC of the Bacteria can be read Directly

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MIC on a strip

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
5-Jan-06 Chiang Mai University 60

Antibiotic Sensitivity testing can be done with automation

Dr.T.V.Rao MD


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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 a staff member  Each laboratory should have

What is the Role of Microbiology Departments

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
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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‟ learning resources for Microbiologists in Developing World
 Email  doctortvrao@gmail.com

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