Antibiotic Sensitivity Testing In

8/7/2019 Antibiotic Sensitivity Testing In

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Dr.T.V.Rao MD

Dr.T.V.Rao MD 1


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Uses of Antibiotic Sensitivity

TestingAntibiotic sensitivity test: A laboratory

test which determines how effective

antibiotic therapy is against a bacterialinfections.

Antibiotic sensitivity testing will controlthe use of Antibiotics in clinical practice

Testing will assist the clinicians in thechoice of drugs for the treatment ofinfections.

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The goal of antimicrobial susceptibility testing is to

predict the in vivo success or failure of antibiotictherapy. Tests are performed in vitro, and measure

the growth response of an isolated organism to aparticular drug or drugs. The tests are performedunder standardized conditions so that the results arereproducible. The test results should be used toguide antibiotic choice. The results of antimicrobial

susceptibility testing should be combined withclinical information and experience when selectingthe most appropriate antibiotic for our patients.

Dr.T.V.Rao MD 3

What is the goal of Antibiotic

Sensitivity testing?


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Components of Antibiotic

Sensitivity Testing1.The identification of relevant pathogens in

exudates and body fluids collected from

patients2. Sensitivity tests done to determine the

degree of sensitivity or resistance ofpathogens isolated from patient to an

appropriate range of antimicrobial drugs3. Assay of the concentration of an

administered drug in the blood or body fluidof patient required to control the schedule of

dosage. Dr.T.V.Rao MD 4


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Why Need Continues for TestingAntibiotic Sensitivity

Bacteria have the abilityto develop resistancefollowing repeated or

subclinical (insufficient)doses, so more advancedantibiotics and syntheticantimicrobials arecontinually required to

overcome them.

Antibiotic sensitivitytesting is essential part ofMedical Care

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Dr.T.V.Rao MD 6

IntroductionSusceptibility 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 patternof 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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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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Routine Susceptibility Tests

Disk diffusion(Kirby Bauer)

Broth micro-dilution MIC

NCCLS referencemethod

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) routinelyperformed.


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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 availablein most hospitals

and for which routine testingshould be carried out forevery strain Set 2:the drugs that are tested only: at the special requestof the physician or when the causative organism isresistantto 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 susceptibilitytests(http://w3.whosea.org/)

Set 1 Set 2

Staphylococcus Benzyl penicillinOxacillin

Erythromycin

Tetracycline

Chloramphenicol

GentamicinAmikacin

Co-trimoxazole

Clindamycin

Intestinal Ampicillin

ChloramphenicolCo-trimoxazole

Nalidixic acid

Tetracycline

Norfloxacin

Enterobacteriaceae

Urinary

Sulfonamide

Trimethoprim

Co-trimoxazole

Ampicillin

Nitrofurantoin

Nalidixic acid

Tetracycline

Norfloxacin

Chloramphenicol

Gentamicin

Blood and tissues Ampicillin

ChloramphenicolCotrimoxazole

Tetracycline

Gentamicin

Cefuroxime

CeftriaxoneCiprofloxacin

Piperacillin

Amikacin

Pseudomonas aeruginosa Piperacillin

Gentamicin

Tobramycin

Amikacin


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Diffusion method

Put a filter disc, or a porous cup/a bottomless cylinder

containing measured quantity of drugs on the a solidmedium that has been seeded with test bacteria

Dilution method

vary amount of antimicrobial substances incorporatedinto liquid or solid media

followed by inoculation of test bacteria

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Antimicrobial Susceptibility

Testing


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Susceptibility Testing Methods

InoculateMH plate

Place diskson agar plate

Incubate plate18-24 hr, 35 CMeasure andrecord zone ofinhibition around

each disk


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Dr.T.V.Rao MD 15

Diffusion MethodDisc diffusion method : The Kirby-Bauer testAntibiotic-impregnated filter disc*

Susceptibility test against more than oneantibiotics by measuring size of inhibition zone

1949: Bondi and colleagues paper disks 1966: Kirby, Bauer, Sherris, and Tuck filter

paper disksDemonstrated that the qualitative results of

filter disk diffusion assay correlated well withquantitative results from MIC tests


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Dr.T.V.Rao MD 16

Disc Diffusion MethodProcedure(Modified Kirby-Bauer method: National

Committee for Clinical Laboratory Standards. NCCLS)

Prepareapproximately.

10

8

CFU/ml bacterial inoculum ina saline or tryptic soy broth tube(TSB) or Mueller-Hintonbroth (5ml) Pick 3-5 isolated colonies from plate

Adjust the turbidity tothesame as the McFarland No.

0.5 standard.* Streak the swabon 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 plateSelect the Colonies from Pure Isolates

Reflected light

Transmitted light


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Disk Diffusion

Test

Select coloniesPrepare inoculumsuspension

Prepare inoculumsuspension

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Prepare the Material for

Inoculation

Standardize inoculumSuspension as per Mac farlandstandard

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

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Disc Diffusion Method Place the

appropriate drug-impregnated disc onthe surface of theinoculated agar plate

Invert the plates andincubate them at35oC, o/n(18-24 h)

Measure thediameters ofinhibition zone inmm

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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 ofinhibition

When beta-lactamase producing Streptococci are tested,zone of inhibition are produced with a heaped-up,clearly defined edge, regardless of the size of theinhibition zone, they should be reported as resistant


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Dr.T.V.Rao MD 26

Look at the Charts for establishingthe zones of Sensitivity

The zone sizes are lookedup on a standardized

chart to give a result ofsensitive, resistant, orintermediate. Manycharts have acorresponding column

that also gives the MIC(minimal inhibitoryconcentration) for thatdrug.
http://www.rlc.dcccd.edu/mathsci/reynolds/micro/lab_manual/glossary.htmlhttp://www.rlc.dcccd.edu/mathsci/reynolds/micro/lab_manual/glossary.html


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Dr.T.V.Rao MD 27

Disc Diffusion MethodReporting the Results

Interpretation of results

By comparing with the diameters withstandard tables

Susceptible

Intermediate susceptible

Low toxic antibiotics: Moderate susceptible

High toxic antibiotics: buffer zone btw resistantand 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 lightinoculum and vice versa

If after application of disc, theplate is kept for longer time atroom temperature, small zonesmay form

Larger zones are seen withtemperatures < 35oC

Ideal 16-18 hours; less timedoes not give reliable results


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Factors Affecting Size of Zone ofInhibition

Size of the plate

Depth of the agarmedium(4 mm)

Proper spacing ofthe discs (2.5 cm)

Smaller platesaccommodate lessnumber of discs

Thin media yieldexcessively largeinhibition zones and viceversa

Avoids overlapping ofzones


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Dr.T.V.Rao MD 30

Factors Affecting Size of Zone ofInhibition

Potency of antibioticdiscs

Composition ofmedium

Acidic pH of medium

Alkaline pH of

medium Reading of zones

Deterioration in contents leadsto reduced size

Affects rate of growth,diffusion of antibiotics andactivity of antibiotics

Tetracycline, novobiocin,methicillin zones are larger

Aminoglycosides,erythromycin zones are larger

Subjective errors indetermining the clear edge


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Dr.T.V.Rao MD 31

Quality Assurance in AntibioticSusceptibility Testing

Visit - WHO-Regional Office for South East Asiawebsite

Medium: Mueller-Hinton agar plates Enterococcus faecalis (ATCC 29212 or 33l86) and a disc of

co-trimoxazole 20 mm in diameter of the inhibitionzone

Procedure: Modified Kirby-Bauer method

recommended by National Committee on ClinicalLaboratory Services (NCCLS)

Susceptibility test with quality control strains


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Dr.T.V.Rao MD 32

Quality Assurance in AntibioticSusceptibility 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)

Pseudomonasaeruginosa (ATCC2785 )


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Dr.T.V.Rao MD 33

Quality Assurance in AntibioticSusceptibility 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 -20oC Use Mueller-Hinton medium for antibiotic

sensitivity determination

Use appropriate control cultures

Use standard methodology for the test


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Modified Methods in Disc diffusion for

Antibiotic sensitivity testing to be used for

detections of following bacterial isolates1 MRSA

2 ESBL

3 Enterobacteriaceae and Gram negativebacteria and Carbapenems resistant usingModified Hodge test

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Need for Modified Methods


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Dilution MethodMinimum Inhibition Concentration (MIC) The lowest concentration of antimicrobial agent that

inhibitsbacterial growth/ multiplication

Minimum Bactericidal Concentration (MBC) orMinimum Lethal Concentration (MLC)

The lowest concentration ofantimicrobial agent that allows less than0.1% 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 MethodProcedure

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 andconcentration ofantibiotic (mg/l)

Controls:

C1 = No antibiotic, checkviability on agar platesimmediatelyC2 = 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 2

Record visual turbidity

Subculture non-turbid tubes

to agar plates (use 0.01 mlstandard 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 platesubcultured with 0.01 ml 500*0.01 = 5 CFU


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Dr.T.V.Rao MD 42

Broth Dilution Method are

Technically DifficultDisadvantages :

Only oneantibiotic & oneorganism can betested each time

Time-consuming

Solutions??Agar dilution

method

Disc diffusionmethod

Micro broth dilutionmethod

Mi b th Dil ti


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Micro broth DilutionMethod

Micro dilution plates: Micro dilution/ Micro broth dilutions

96 wells/ plate: simultaneously performedwith many tests organisms/ specimens, less

reagent requiredManually preparedCommercially prepared Frozen or Dried/ lyophilized

Consistent performance but high costMay suffer from degradation of antibiotic during

shipping and storage


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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

MIC32 ug/ml


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Minimal inhibitory concentration

The lowestconcentration ofantimicrobial agentthat inhibits thegrowth of abacterium

Interpret:

Susceptible Intermediate

Resistant


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Clinical Conditions when MICs are

Useful

Endocarditis

Meningitis

SepticemiaOsteomyelitis

Immunosuppressed patients (HIV, cancer,

etc.)Prosthetic devices

Patients not responding despite S Reports

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I l P i


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Inoculum PreparationMIC Testing

(NCCLS Reference Method)

Standardizeinoculum

suspensionFinal 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 dilutionMIC tray

Prepare inoculum

suspension

Dil & i i l


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Dilute & mix inoculumsuspension


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Pour inoculum into reservoir andinoculate MIC tray


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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 perplate - may not reveal contaminants if no isolatedcolonies)

Examine before reading MIC (usually at 16-20 h)

Re-incubate if Antibiograms questionable


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- +

64

32

16

8

4

2

1

>64

0.5

Read

MICs

>64

Th di t t h i


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Dr.T.V.Rao MD 55

The gradient technique,

EtestEtest is a well established

AST method inmicrobiology laboratories

around the world. The Etesttechnique comprises apredefined gradient ofantibiotic concentrations ona plastic strip, and can beused to determine theMinimum Inhibitory

Concentration (MIC) ofantibiotics, antifungal agentsand antimycobacterialagents.


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E test MIC Reports are helpful inCritical management decisions

Quantitative MIC datais a prerequisite for the

management of criticalinfections, includingsepsis, especiallyamong critical carepatients. Etest isparticularly valuable insuch situations, whenon-scale MICs areneeded for treatment

decisions.


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Antimicrobial Gradient Testing

E-test

Read platesafter

recommendedIncubation

Read MICwhere elipse

intersectsscale


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MIC of the Bacteria can be readDirectly


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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 patients

serum = MIC*SIT The Serum Inhibitory Titer (SIT)

The highest dilution of patients serum that inhibitbacteria

To determine the ability of drug in the patientsserum to kill bacteria

The Serum Bactericidal Level (SBL) The lowest dilution of patients serum that kills bacteria

Technically Demanding


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Antibiotic Sensitivity testingcan be done with automation

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VITEK 2 Automates Reportingof Resistance

Integrated in the VITEK 2system is the AdvancedExpert System (AES), asoftware which validatesand interprets susceptibilitytest results, and detectsantibiotic resistancemechanisms. The AESExpert System is the mostdeveloped software system

in this field, and is capableof identifying evenemerging and low-levelresistance.


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Each laboratory should have a staff member

with the time, interest, and expertise to provideleadership in antibiotic testing and resistance.This person would read relevant publications,network with other laboratories, and evaluatepotentially useful tests to detect new forms of

resistance before new CLSI-recommendedtests become available

- Ken Thomson, Emerging Infect. Dis., 2001

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What is the Role of

Microbiology Departments


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1Usanee Anukool (Ph.D.) Clinical Microbiology,AMS,Chiang Mai University2National Committee For Clinical Laboratory Standards. 1998.

NCCLS document M100 - S8 . Performance Standards forAntimicrobial Susceptibility Testing. 8th edition, NCCLS, Waynae,Pa.

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References


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Antibiotic Sensitivity Testing In