kirby-bauer disc diffusion method antibiotic susceptibility testing skill based learning

8/6/2019 KIRBY-BAUER DISC DIFFUSION METHOD ANTIBIOTIC SUSCEPTIBILITY TESTING SKILL BASED LEARNING

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Dr.T.V.Rao MD

KIRBY-BAUER DISC DIFFUSION METHOD

ANTIBIOTIC SUSCEPTIBILITY TESTING

SKILL BASED LEARNING

DR.T.V.RAO MD 1


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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

ofAntibiotics in clinical practice

Testing will assist the clinicians in the choice

of drugs for the treatment of infections.

DR.T.V.RAO MD 2


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ANTIBIOTIC SUSCEPTIBILITY

testing has become a veryessential step for properly treating

infectious diseases and

monitoring antimicrobial

resistance in various pathogens.The choice of antibiotic needs to be

made taking into consideration the

susceptibility profile of the pathogen,

pharmacology of the antibiotic, the

need for antibiotic therapy, and its

cost effectiveness

ANTIBIOTIC SUSCEPTIBILITY IS

IMPORTANT IN PATIENT CARE

DR.T.V.RAO MD 3


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INDICATIONS FOR ROUTINE SUSCEPTIBILITY

TESTING

Susceptibility test may be performed in the

clinical laboratory for two main purposes:

To guide the clinician in selecting the bestantimicrobial agent for an individual patient

To accumulate epidemiological information on

the resistance of microorganisms of public

health importance within the community.

DR.T.V.RAO MD 4


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Susceptibility tests should never

be performed on contaminants or

commensals belonging to the

normal flora, or on other

organisms that have no causal

relationship to the infectious

process. These should be

carried out only on pure

cultures of organisms

considered to be causing the

infectious process. Theorganisms should also be

identified since not every

microorganism isolated from a

patient with an infection requires

an Antibiograms.

SUSCEPTIBILITY TEST AS A GUIDE FOR

TREATMENT

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Outline susceptibility tests arenot indicated when the causativeorganism belongs to a specieswith predictable susceptibility tospecific drugs. This is the case

forStreptococcus pyogenesand Neisseria meningitides,which are still generallysusceptible to penicillin. Ifresistance of thesemicroorganisms is suspected on

clinical grounds, repres-entativestrains should be submitted to acompetent reference laboratory.

ANTIBIOTIC SENSITIVITY TESTING NOT

NEEDED IN ALL ISOLATES

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Routine susceptibility tests on major

pathogens (e.g. S.typhi,shigellae) are useful as part ofa comprehensive programme ofsurveillance of enteric infections.

These are essential for informingthe physician of the emergenceof resistant strains(chloramphenicol resistantS.typhi, co-trimoxazoleresistant and ampicillin

resistant shigellae) and indicatea need to modify standardtreatment schemes.

SUSCEPTIBILITY TEST AS AN EPIDEMIOLOGICAL

TOOL

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Continued surveillance of the results

of routine susceptibility tests is an

excellent source of information on

the prevalence of resistant

staphylococci and Gram-negative

bacilli that may be responsible forcross-infections in the hospital.

Periodic reporting of the

susceptibility pattern of the prevalent

strains is an invaluable aid to

forming a sound policy on antibiotic

usage in the hospital by restrictionand/or rotation of life-saving drugs,

such as the aminoglycosides and

cephalosporins.

SUSCEPTIBILITY TEST AS AN

EPIDEMIOLOGICAL TOOL

DR.T.V.RAO MD 8


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OTHER ESSENTIAL INGREDIENTS

The Commitment Value of antibiotic QC

For the patient

The laboratory

The Hands and Heads

Skilled personnel

The media

The incubator

The bacterial isolate The antibiotics

The standard operating procedures (SOP)

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The choice of drugs used in a

routine Antibiograms is governed by

considerations ofthe antibacterial

spectrum of the drugs, their

pharmacokinetic properties,

toxicity, efficacy, and availability, aswell as their cost to both the patient

and the community. Among the

many antibacterial agents that could

be used to treat a patient infected

with a given organism, only a limited

number of carefully selected drugsshould be included in the

susceptibility test.

CHOICE OF DRUGS

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In the standardized method , the

inoculum is prepared from

colonies on a primary culture

plate or from a pure culture. This

is called an "indirectsensitivity test". In certaincases, where a rapid answer is

important, the standardized

inoculum may be replaced by the

pathological specimen itself, e.g.urine, a positive blood culture, or

a swab of pus

DIRECT VERSUS INDIRECT SUSCEPTIBILITY

TESTS

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DIRECT SUSCEPTIBILITY TESTING

For urine specimens, a microscopic examination of the sediment should first

be made in order to see if there is evidence of infection, i.e. the presence of

pus cells and/or organisms. The urine may then be used as the inoculum in

the standard test. Likewise, susceptibility tests may be performed on

incubated blood cultures showing evidence of bacterial growth, or a swab of

pus may be used as a direct inoculum, when a Gram stained smear shows

the presence of large numbers of a single type of organism. This is called a

"direct susceptibility test"; its advantage over the indirect test is that a result

is obtained 24 hours earlier. The disadvantage is that the inoculum cannot be

properly controlled. When the susceptibility plate shows too light or too

heavy growth, or a mixed culture, the results should be interpreted withcaution or the test repeated on pure cultures.

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Staphylococcus

Benzyl penicillin

Oxacillin

ErythromycinTetracycline

Chloramphenicol

Gentamicin

AmikacinCo-trimoxazole

Clindamycin

-

STAPHYLOCOCCUS

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EnterobacteriaceaeUrinary

SulfonamideTrimethoprim

Co-trimoxazoleAmpicillinNitrofurantoinNalidixic acidTetracycline

NorfloxacinChloramphenicolGentamicin

ENTEROBACTERIACEAE

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Blood and tissues

AmpicillinChloramphenicolCo-trimoxazoleTetracycline

CefalotinGentamicin

CefuroximeCeftriaxoneCiprofloxacin

PiperacillinAmikacin

* Testing as per nature of isolate

*BLOOD AND TISSUES

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Pseudomonas aeruginosa

Piperacillin

GentamicinTobramycin

Amikacin

PSEUDOMONAS AERUGINOSA

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DR.T.V.RAO MD 17

BASIC SETS OF DRUGS FOR ROUTINESUSCEPTIBILITY TESTS

(HTTP://W3.WHOSEA.ORG/)

Set 1 Set 2Staphylococcus Benzyl penicillin

Oxacillin

Erythromycin

Tetracycline

Chloramphenicol

Gentamicin

Amikacin

Co-trimoxazole

Clindamycin

Intestinal AmpicillinChloramphenicol

Co-trimoxazole

Nalidixic acidTetracycline

Norfloxacin

Enterobacteriaceae

Urinary

Sulfonamide

Trimethoprim

Co-trimoxazoleAmpicillin

Nitrofurantoin

Nalidixic acid

Tetracycline

Norfloxacin

Chloramphenicol

Gentamicin

Blood and tissues AmpicillinChloramphenicol

Cotrimoxazole

TetracyclineGentamicin

Cefuroxime

Ceftriaxone

Ciprofloxacin

PiperacillinAmikacin

Pseudomonas aeruginosa Piperacillin

Gentamicin

Tobramycin

Amikacin


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GENERAL PRINCIPLES OF ANTIMICROBIAL

SUSCEPTIBILITY TESTING

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Antimicrobial

susceptibility tests

measure the ability of

an antibiotic or otherantimicrobial agent to

inhibit bacterial growth

in vitro. This ability may

be estimated by eitherthe dilution method or

the diffusion method.

ANTIMICROBIAL SUSCEPTIBILITY

TESTS

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Forquantitative estimates ofantibiotic activity, dilutions of theantibiotic may be incorporated intobroth or agar medium, which is theninoculated with the test organism.The lowest concentration that

prevents growth after overnightincubation is known as the minimuminhibitory concentration (MIC) of theagent. The MIC value is thencompared with knownconcentrations of the drug

obtainable in the serum and in otherbody fluids to assess the likelyclinical response.

THE DILUTION METHOD

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Paper discs impregnated with a

defined quantity of antimicrobial

agent are placed on agar

medium uniformly seeded with

the test organism. A

concentration gradient of theantibiotic forms by diffusion from

the disc and the growth of the

test organism is inhibited at a

distance from the disc that is

related among other factors tothe susceptibility of the

organism.

THE KIRBY-BAUER DISC DIFFUSION

METHOD

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The recommended method for

intermediate and peripheral

laboratories is the modified Kirby-

Bauer method, the methodology :

This method has been

recommended by NationalCommittee on Clinical Laboratory

Services (NCCLS-USA)

Subcommittee on Antimicrobial

Susceptibility Testing. This is the

most thoroughly described disc

diffusion method for whichinterpretive standards have been

developed and which is supported

by laboratory and clinical data.

KIRBY-BAUER METHOD

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THE MODIFIED KIRBY-BAUER

METHOD

Mueller-Hinton agar

1. Mueller-Hinton agar should be prepared from a dehydrated base

according to the manufacturer's recommendations. The medium should be

such that control zone sizes within the standard limits are produced. It is

important not to overheat the medium.

2. Cool the medium to 45-50oC and pour into the plates. Allow to set on a

level surface, to a depth of approximately 4 mm. A 9 cm diameter plate

requires approximately 25 mL of the medium.

3. When the agar has solidified, dry the plates for immediate use for l0-30minutes at 36oC by placing them in an upright position in the incubator with

the lids tilted.

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THE MODIFIED KIRBY-BAUER METHOD

4. Any unused plates may be stored in a plastic bag, which

should be sealed and placed in the refrigerator. Plates stored

in this way can be kept for 2 weeks.

To ensure that the zone diameters are sufficiently reliable fortesting susceptibility to sulfonamides and co-trimoxazole,

Mueller-Hinton agar must have low concentrations of the

inhibitors thymidine and thymine. Each new lot of Mueller-Hinton

agar should therefore be tested with a control strain of

Enterococcus faecalis (ATCC 29212 or 33l86) and a disc ofco-trimoxazole. A satisfactory lot of medium will give a distinct

inhibition zone of 20 mm or more that is essentially free of hazy

growth or fine colonies.

DR.T.V.RAO MD 24

EXAMINING PURITY OF PLATE


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DR.T.V.RAO MD 25

EXAMINING PURITY OF PLATESELECT THE COLONIES FROM PURE ISOLATES

Reflecte

d light

Transmitted light


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To ensure that the zone diameters

are sufficiently reliable for testing

susceptibility to sulfonamides and

co-trimoxazole, Mueller-Hinton agar

must have low concentrations of the

inhibitors thymidine and thymine.Each new lot of Mueller-Hinton agar

should therefore be tested with a

control strain ofEnterococcus

faecalis (ATCC 29212 or 33l86)

and a disc of co-trimoxazole. A

satisfactory lot of medium will give adistinct inhibition zone of20 mm or

more that is essentially free of

hazy growth or fine colonies .

QUALITY OF THE MUELLER HINTON GIVES

OPTIMAL RESULTS

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Any commercially available discs with

the proper diameter and potency can

be used. Stocks of antibiotic discs

should preferably be kept at -20oC;

the freezer compartment of a home

refrigerator is convenient. A small

working supply of discs can be kept inthe refrigerator for up to 1 month. On

removal from the refrigerator, the

containers should be left at room

temperature for about l hour to allow

the temperature to equilibrate. This

procedure reduces the amount ofcondensation that occurs when warm

air reaches the cold container.

STORING OF ANTIBIOTIC DISCS

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Stored and handledcorrectly

Refrigeration taken out 1hour before use

Expiry dates noted Discs at room

temperature before use

Avoid condensation

Placing of discs within 15minutes of swabbing

CARE OF THE ANTIBIOTIC TESTING

DISCS

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Prepare the turbidity standard by

pouring 0.6 mL of a 1% (10 g/L)

solution of barium chloride

dihydrate into a l00-mL

graduated cylinder, and filling to

l00 mL with 1% (10 mL/L) sulfuricacid. The turbidity standard

solution should be placed in a

tube identical to the one used for

the broth sample. It can be

stored in the dark at roomtemperature for 6 months,

provided it is sealed to prevent

evaporation.

FOLLOW THE TURBIDITY STANDARDS

TO SUIT THE SITUATION

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Stock of cotton wool

swabs on wooden

applicator sticks should

be prepared. These canbe sterilized in tins,

culture tubes, or on

paper, either in the

autoclave or by dryheat.

USE OF SWABS AND PREPARATION

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PROCEDURE

DR.T.V.RAO MD 31

Kirby-Bauer disc diffusion method


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To prepare the inoculum from

a primary culture plate, touch

with a loop the tops of each

of 3-5 colonies of similar

appearance of the organismto be tested.

When the inoculum has to be

made from a pure culture, a

loopful of the confluent growthis similarly suspended in

saline, or peptone water.

PICKING UP THE COLONIES FROM PURE

ISOLATES

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Compare the tube with the turbidity

standard and adjust the density of the

test suspension to that of the standard

by adding more bacteria or more

sterile saline. Proper adjustment to the

turbidity of the inoculum is essential to

ensure that the resulting lawn ofgrowth is confluent or almost

confluent.

Inoculate the plates by dipping a sterile

swab into the inoculum. Remove excess

inoculum by pressing and rotating theswabs firmly against the side of the

tube above the level of the liquid.

PREPARE THE OPTIMAL INOCULUM

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Streak the swab all over the surfaceof the medium three times, rotating theplate through an angle of 60o aftereach application. Finally, pass theswab round the edge of the agarsurface. Leave the inoculum to dry for

a few minutes at room temperaturewith the lid closed. The antibiotic discsmay be placed on the inoculatedplates using a pair of sterile forceps.

A sterile needle tip may also beused to place the antibiotic discs onthe plate. Alternatively, an antibiotic

disc dispenser can be used to applythe discs to the inoculated plate.

PREPARE THE LAWN CULTURE OF THE

ISOLATE

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A maximum of seven discs can be

placed on a 9-10 cm diameter plate.

Six discs may be spaced evenly,

approximately 15 mm from the edge of

the plate, and 1 disc placed in the

center of the plate. Each disc should

be gently pressed down to ensureeven contact with the medium.

The plates should be placed in an

incubator at 35oC within 30 minutes of

preparation. Temperatures above

35oC invalidate results for

oxacillin/methicillin.

PLACEMENT OF THE ANTIBIOTIC DISCS

DR.T.V.RAO MD 35

STACKING OF PLATES


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STACKING OF PLATESNO MORE THAN 5 PLATES HIGH

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INCUBATION

Knowledge of requirements for

incubation

Ambient air at 350C for non

fastidious aerobic and facultativeanaerobic bacteria

Timing generally 16-18 hours

24 hours forS.aureus and

enterococci

5% CO2 forHaemophilus

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Do not incubate in anatmosphere of carbon dioxide.

After overnight incubation,the diameter of each zone

(including the diameter of thedisc) should be measuredand recorded in mm. Theresults should then beinterpreted according to thecritical diameters bycomparing them with standardtables.

MEASURING THE ZONES OF INHIBITION

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The measurements can be made

with a ruler on the under surface

of the plate without opening the

lid.

The endpoint of inhibition isjudged by the naked eye at the

edge where the growth starts,

but there are three exceptions.

With sulfonamides and co-

trimoxazole, slight growth occurswithin the inhibition zone; such

growth should be ignored.

MEASURE ZONE WITH RULER FOR OPTIMAL

REPORTING

DR.T.V.RAO MD 39


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When b-lactamase producingstaphylococci are tested againstpenicillin, zones of inhibition areproduced with a heaped-up, clearlydefined edge; these are readilyrecognizable when compared with the

sensitive control, and regardless of thesize of the zone of inhibition, theyshould be reported as resistant.

Certain Proteus species may swarminto the area of inhibition around someantibiotics, but the zone of inhibition is

usually clearly outlined and the thin layerof swarming growth should be ignored.

OBSERVATION VARY ACCORDING TO NATURE

OF ISOLATE

DR.T.V.RAO MD 40

CLINICAL DEFINITIONS OF TERMS


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CLINICAL DEFINITIONS OF TERMS

RESISTANT AND SUSCEPTIBLE: THE THREE-

CATEGORY SYSTEM

The result of the susceptibility test, as reported to the clinician,

is the classification of the microorganism in one of two or more

categories of susceptibility. The simplest system comprises only

two categories, susceptible and resistant. This classification,

although offering many advantages for statistical andepidemiological purposes, is too inflexible for the clinician to

use. Therefore, a three-category classification is often adopted.

The Kirby-Bauer method recognizes three categories of

susceptibility and it is important that both the clinician and thelaboratory worker understand the exact definitions and the

clinical significance of these categories.

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An organism is called"susceptible" to adrug when theinfection caused by itis likely to respond totreatment with thisdrug at the

recommendeddosage.

SUSCEPTIBLE:

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This term covers two situations. It is

applicable to strains that are

"moderately susceptible" to an

antibiotic that can be used for

treatment at a higher dosage

because of its low toxicity or

because the antibiotic is

concentrated in the focus of

infection (e.g. urine). The term also

applies to those strains that are

susceptible to a more toxic antibiotic

that cannot be used at a higherdosage. In this situation this

category serves as a buffer zone

between susceptible and resistant.

INTERMEDIATE SUSCEPTIBILITY

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This term implies

that the organism is

expected not to

respond to a givendrug, irrespective of

the dosage and of

the location of theinfection.

RESISTANT:

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READ THE RESULTS WITH PRECISION

TransmittedLight

DR.T.V.RAO MD 45


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NEED FOR QUALITY CONTROL IN

SUSCEPTIBILITY TEST

The final result of a disc diffusion test is influenced by a

large number of variables. Some of the factors, such as

the inoculum density and the incubation temperature,

are easy to control, but a laboratory rarely knows theexact composition of a commercial medium or the

batch-to-batch variations in its quality, and it cannot

take for granted the antimicrobial content of the discs.

The results of the test must, therefore, be monitoredconstantly by a quality control programme which should

be considered part of the procedure itself.

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NEED FOR QUALITY CONTROL IN SUSCEPTIBILITY

TEST

DR.T.V.RAO MD 47

The precision and accuracy of the test are controlled by the

parallel use of a set of control strains, with known susceptibility

to the antimicrobial agents. These quality control strains are

tested using exactly the same procedure as for the test

organisms. The zone sizes shown by the control organismsshould fall within the range of diameters standardised. When

results regularly fall outside this range, they should be regarded

as evidence that a technical error has been introduced into the

test, or that the reagents are at fault. Each reagent and eachstep in the test should then be investigated until the cause of

the error has been found and eliminated.

STANDARD PROCEDURE FOR QUALITY


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The quality controlprogramme should usestandard reference strains ofbacteria that are tested inparallel with the clinicalculture. They shouldpreferably be run every weekor with every fifth batch oftests, and in addition, everytime that a new batch of

Mueller Hinton agar

or a new batch ofdiscs is used.

Q

CONTROL

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POSSIBLE ERRORS

Inoculum too light

Antibiotic too potent

QC strain mutated

Agar too thin

Incorrect recording of

results

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Staphylococcus aureus(ATCC 25923)Escherichia coli (ATCC25922)

Pseudomonas aeruginosa(ATCC 27853)

culture for day-to-day use should begrown on slants of nutrient agar(Tryptic soya agar is convenient)and stored in the refrigerator. These

should be subcultured onto freshslants every 2 weeks.

STANDARD STRAINS

DR.T.V.RAO MD 50

FREQUENCY OF QUALITY CONTROL


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FREQUENCY OF QUALITY CONTROL

TESTING

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 sensitivitydetermination

Use appropriate control cultures

Use standard methodology for the test

Use coded strains from time to time for internal qualitycontrol

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FREQUENCY OF QUALITY CONTROL TESTING

Keep the antibiotic discs at room temperature for one hour before use

Incubate the sensitivity plates for 16-18 hours before reporting

Incubate the sensitivity plates at 35oC

Space the antibiotic discs properly to avoid overlapping of inhibition

zone Use inoculum size that produces near confluent growth

Ensure even contact of the antibiotic disc with the inoculatedmedium

Measure zone sizes precisely Interpret zone sizes by referring to standard charts

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The zone size produced by an

antimicrobial agent indicates its

activity against the organism.

However, zone sizes of two

agents to which the organism is

sensitive are not comparable andshould not give an erroneous

impression that the test

organism is more

sensitive to the drug

which has yielded abigger zone size.

COMPARING TWO DRUG SUSCEPTIBILITIES

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WHEN THINGS GO WRONG

Questions to ask?

Is the procedure correct?

Check test materials including test strains

Check equipment

Fridges

Incubators

Freezers

Review technique of personnel

Culture of general QC in lab essential

THE HANDS AND HEADS OF


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THE HANDS AND HEADS OF

PERSONNEL ARE IMPORTANT

Essential that everyone is awareof the importance of QC

Training of personnel in correcttechnique

Storage of discs

Preparation of a standard inoculum

Swabbing of plates

Choice and storage of media

Timing and methods of incubation ofplates

Measurement of zone sizes

Recording of results

Preparing a standard inoculumDR.T.V.RAO MD 55


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INVESTIGATION OF ERRORS

Quality of media should be investigated

pH will affect macrolides, tertracyclines and aminoglycoside

In this case the pH was too low acidic

Ideal pH 7.2-7.4 TMP-SMX is affected by the amount of thymidine in

media

This QC may indicate the presence of excess thymidine in the

agar which will allow the bacteria to bypass the inhibitoryeffects of TMP-SMX

Corrective action should be taken in house or with themanufacturer and QC repeated with a new batch


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AST QC needs a culture ofgeneral QC in thelaboratory

Systems for performing,recording andtroubleshooting should bedocumented in writing

Any errors should beinvestigated timeously andsystematically

Results can then bereported with confidenceand permit appropriate andsafe antimicrobial use

ROLE OF LABORATORY MANAGERS

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DR.T.V.RAO MD 58

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kirby-bauer disc diffusion method antibiotic susceptibility testing skill based learning

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