Dr.T.V.Rao MD 1

ANTIBIOTIC SENSITIVITY TESTINGSKILL BASED LEARNING

Dr.T.V.Rao MD

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.

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?

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.

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

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)

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

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) ?

Dr.T.V.Rao MD 9

Routine Susceptibility

Tests Disk diffusion

(Kirby Bauer) Broth micro-

dilution MIC NCCLS

reference method

Etest

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.

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

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

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

Susceptibility Testing Methods

InoculateMH plate

Place diskson agar plate

Incubate plate18-24 hr, 35 CMeasure and record zone of inhibition around each disk

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

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

Dr.T.V.Rao MD 17

Examining purity of plateSelect the Colonies from Pure

Isolates

Reflected light

Transmitted light

Dr.T.V.Rao MD 18

Disk Diffusion

Test

Select colonies

Prepare inoculumsuspension

Prepare inoculumsuspension

Dr.T.V.Rao MD 19

Prepare the Material

for Inoculation

Standardize inoculumSuspension as per Mac farland standard

Mix well

Dr.T.V.Rao MD 20

Swab the plate with

optimal sample

Remove sample

Swab plate

Dr.T.V.Rao MD 21

Select the Disks and

Apply

Select disks

Dr.T.V.Rao MD 22

Incubate

Overnight

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

Dr.T.V.Rao MD 24

Read the Results

with Precision

TransmittedLight

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

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.

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

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

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

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

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

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 )

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

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

35

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

Antimicrobial susceptibilitytesting using micro-broth

dilutions

•

•

••••

•• •

•

•

••

96 well microtiter plate

ug/ml64 32 16 8 4 2

37

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

Dr.T.V.Rao MD 38

Creating Dilutions

39

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

40

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

41

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

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

43

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

44

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

45

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

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

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

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)

Dr.T.V.Rao MD 49

Select Micro titration

plate and prepare optimal inoculum

Micro dilution MIC tray

Prepare inoculumsuspension

Dr.T.V.Rao MD 50

Dilute & mix inoculum

suspension

Dr.T.V.Rao MD 51

Pour inoculum into reservoir

and inoculate MIC tray

Dr.T.V.Rao MD 52

Incubate overnight

Do not forget to check the purity of Inoculum

Inoculate purity plate

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

- +

643216 8 4 2 1

>64

0.5

Read MICs

>64

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.

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.

Antimicrobial Gradient

TestingE-test®

Read platesafter

recommendedIncubation

Read MICwhere elipse

intersectsscale

Dr.T.V.Rao MD 58

MIC of the Bacteria can be

read Directly

MIC on a stripabbiodisk.com

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

Dr.T.V.Rao MD 61

Antibiotic Sensitivity

testing can be done with automation

Dr.T.V.Rao MD 62

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.

Dr.T.V.Rao MD 63

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

Dr.T.V.Rao MD 64

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

Dr.T.V.Rao MD 65

For Articles of Interest on Antibiotics follow me on

Dr.T.V.Rao MD 66

Created by Dr.T.V.Rao MD for ‘e’

learning resources for Microbiologists in Developing

World Email

doctortvrao@gmail.com