ANTIMICROBIAL SENSITIVITY TESTS

@ Used to select effective drugs for treatment .

@ Not indicated if sensitivity pattern of a pathogen can be predicted.

@ Not performed on commensals or contaminants

@ This misleads physician and patient to receive unnecessary therapy

@ Such therapy leads to side-effects & resistance of pathogens.

@ Used to identify organism if it has a characteristic sensitivity pattern.

LIMITATIONS:1.Measure in vitro not in vivo drug

activity.2.Selection of best drug depends on:

a) Patient clinical condition b) Type and site of infection. c) History of drug hypersensitivity.

3.Drug activity : absorption, diffusion in tissues, metabolism, excretion, toxicity, effect on patient normal flora, are not known by sensitivity.

TECHNIQUES:

Mainly two:

1.Diffusion technique.2.Dilution technique.

DIFFUSION SENSITIVITY TECHNIQUE:

@ Used in routine sensitivity testing.

@ A disc of filter paper is impregnated with a known volume & concentration of a drug & placed on an agar medium inoculated with a test organism.

Control organisms are inoculated:

@ On same plate (Stokes technique).

@ On a separate plate (Kirby-Bauer technique).

Drug diffuses into medium.

@ After an overnight incubation, culture is examined for areas of no growth (inhibition zones) around discs:

1.Sensitive bacteria are inhibited at a distance from disc.

2.Resistant bacteria grow up to the edge of disc.

@ In Stokes technique:

• inhibition zone is compared directly with that of control .

@ In Kirby-Bauer technique:

•zone is measured & compared against a previously prepared scale that correlates zone size with MIC.

@ MIC is the minimum drug concentration required to inhibit bacterial multiplication under standard conditions.

@ It is measured by the dilution sensitivity technique.

@ Inhibition zone increases when MIC decreases.

Inhibition zones vary in size due to:

1.Difference in molecular structures of drugs (larger zones are obtained when drugs diffuse rapidly in medium).

2.When bacterial growth is heavy (zones are smaller, & vice versa) .

3.Factors affecting the medium: (volume, moisture, pH, & constituents).

4.Factors affecting the disc: (drug concentration, storage, & application).

DILUTION SENSITIVITY TECHNIQUE:

@ Performed under conditions:

1.Patient not responding to therapy

2.Patient is immunosuppressed.

@ Measures MIC. @ Measures the minimum

bacterial concentration (MBC)

(The minimum concentration of drug required to kill bacteria).

Technique:

@ Dilutions of drug are added to a medium.

@ A standard inoculum of organism is added.

@ After overnight incubation, MIC is reported .

@ Clinical response is assessed by comparing MIC obtained with already known concentrations of the drug

@ MBC may be determined by subculturing last tube in the dilution series to show visible growth

@ Other tubes should detect no growth on subculture.

@ Dilution techniques require: *good standardization *good control of: inoculum, medium, drugs , incubation time, diluting techniques, reading of

results.

@ MIC may be determined by automated machines.

DRUG ASSAYS:@ Performed to: * check enough drug concentration in a body fluid to give adequate therapy

(e.g.: treatment of endocarditis) * make sure that concentrations of toxic aminoglycosides remain below their toxic levels in pt. serum.

@ Specimens submitted for drug assays are accompanied by:

drug dose, time of administration, time of

collecting the specimen, and if patient is receiving other

treatments.

STOKES DISC DIFFUSION SENSITIVITY TESTING:

@ Has the following advantages:

1.Both test & control strains are inoculated on same plate.

2.Inoculum gives semi-confluent growth

(neither too heavy nor too light).

3. The activity of each disc is controlled.

4. Inhibition zone of test organism is compared directly with that of control.

5. Errors due to too heavy or too light inocula are easily detected.

6. Different media may be used.

REQUIREMENTS:

1) SENSITIVITY TESTING AGAR: @ Best is Mueller - Hinton agar.

@ Depth of medium is 4mm (25

ml) @ Pour plates on a level surface.

@ Too thin & too thick media

give false inhibition zones. @ Plates are stored in plastic

bags at 2 - 8°C for up to 2 weeks.

@ Before use dry plates with lids slightly open for ½ hr at 37°C.

@ 5% blood is added to M-H agar to test for fastidious organisms (Neisseria, Haemophilus,

Streptococcus).

Factors affecting antimicrobial sensitivity

1. Media containing substances inhibiting action of aminoglycosides, tetracyclines, trimethoprim, e.g: the substance thymidine.

2.pH of media: False large zones are formed if medium is acidic (tetracycline), or false small zones if medium is alkaline (aminoglycosides).Fermentable sugars are not added to

medium to avoid production of acid and change of pH.

2) ANTIMICROBIAL DISCS:

@ To select drugs for sensitivity, consult clinicians.

@ Drug list must be limited & reviewed at regular intervals.

@ If resistance developed, one member of each drug group is selected.

Other factors include: * Manufacturer instructions regarding

discs: store temp., expiry date, etc. are followed

* Bring discs to room-temp. one hour before use.

* Do not expose discs to sunlight.

* Quality control of discs essential.

* Avoid dryness & heat that decrease control zone size.

3) CONTROL STRAINS: @ Selected according to :

1.Site of infection in patient.

2.Drug concentration at this site.

3.Strain must respond to treatment with normal doses .

4.Strain must grow at same rate as test organism.

Recommended control strains:

1. S.aureus – Oxford strain (NCTC 6571): Used for all except polymyxins, & for pathogens of all specimens except urine.

2. E.coli – NCTC 10418 . Used for all drugs against pathogens from urine.

3. Ps.aeruginosa – NCTC 10662 : Used for controlling all drugs against Ps.

Precautions for control strains are :

1. Strain is cultivated on N.A. slopes.

2. Strains are stored in dark at room temp.(20-28°C).

3. Subculture is made every 3 – 6 months

4. Each week, a nutrient broth or agar culture is made & stored at 2 – 8°C. & from this culture, suspensions are

prepared for daily use.

4) TURBIDITY (OPACITY) STANDARD

@ It is a standard of barium chloride for matching turbidity of test & control strains inocula.

@ Turbidity of standard is equivalent to an overnight broth culture.

@ After matching , don’t incubate test or control strains for two hrs.

Preparation of Turbidity Standard:

1. Add I ml of H2SO4 to 99 ml water to make 1% H 2SO4.

2. Dissolve 2.35g barium chloride in 200 ml water .

3. Mix 0.5 ml barium chloride solution to 99.5 ml H2SO4 solution

4. Transfer turbid solution to screw-cap bottle of same type as that used to prepare test & control strain suspension

5. Store turbidity standard sealed in dark at room temp. for 6 months.

INDIRECT SENSITIVITY TESTING:

@ Indirect (Secondary) test: Inoculum is a pure culture.@ Direct (primary) test: Inoculum is a specimen.

METHODS OF INDIRECT TESTING:

@ Apply Stokes technique as

follows:1. Emulsify colonies of organism in

Muller-Hinton broth.

2. Match turbidity developed against standard turbidity .

* To match, view against a printed card

* No incubation.

3. Using a sterile a 4mm loop , apply the organism suspension to center of sensitivity plate.

@ Using a sterile swab, spread inoculum across the center third of the plate.

Control

Control

Test

@ Do not use same swab for both application and spreading.

@ Judge your turbidity with turbidity standard, not by naked eye.

4. Similarly, inoculate the broth culture of control strain across the upper and lower thirds of plate, leaving 5 mm. on each side of test organism. @ Control suspension must be

standardized against the standard.

5. Allow the inocula to dry for few minutes, with the Petri dish closed.

6. Place antibiotic discs (after warming to room temp.) between test and control inocula.

Press disc down a little, and do not move once it is placed.

7. After ½ hr. incubate at 37°C . @ For methicillin , incubate at

35°C.

8. Read test when : @ Growth of both test and control

strains is not too heavy or too light.

@ Control inhibition zones measure 8-15 mm. radius.

* Measurement is from edge of disc to edge of zone.

@ Using an electric rotary inoculator, test strain may be inoculated in a ring around the control strain.

@ If growth of test & control strains is not semi confluent, sensitivity test is repeated.

INTERPRETATION OF RESULTS:

Test is reported: sensitive, intermediate, & resistant:a) Sensitive: Test zone is: @ wider than control zone @ or equal to control zone @ or not than 3 mm smaller of control zone.

b)Intermediate:Test zone is: @ more than 3 mm smaller of control zone but not less than 3 mm in diameter.c) Resistant: Test zone is: @ 2 mm or less. @ No zone of inhibition

1. Intermediate zone drugs must be prescribed in high doses to cure infection, or when drug is concentrated at site of infection, e.g.: UTI.

2. With sulphonamides & trimethoprim, slight growth may occur within inhibition zone. This is due to presence of inhibitors (thymidine), and it must be ignored.

3. Strains are considered resistant if: @ Growth is heaped-up at zone edge

without gradual fading up towards disc (penicillin-resistant Staph.) @ Large colonies are seen growing within inhibition zone

(Staph.aureus)

4. To test for sensitivity of co- trimoxazole , test for sensitivity of sulphamoxazole & trimethoprim separated, using individual discs

each.

5. Colistin & polymyxin give smaller zones of inhibition because of their large molecular size. Hence control zone must be at least 3-4mm..

6. If Proteus swarms across its inhibition zone, no problem, the zone is clear.

7. Check your discs daily for any decrease in zone size resulting from drug deterioration.

DIRECT SENSITIVITY TESTING:

@ Performed when :1. Gram smear stain showed large number of one type of organism.2. To obtain a presumptive result

for serious cases 3. For urine, pus, pos. blood

cultures,

4. To isolate & identify a pathogen or

to detect a resistant strain.

@ Sensitivity plate must not replace routine culture plate.

@ Blood is added to Muller Hinton agar to be used for direct sensitivity

@ Procedure for direct sensitivity is same as for indirect sensitivity .

@ Result of direct sensitivity must be confirmed by indirect sensitivity .

@ Do not report direct sensitivity result if:

* Growth of bacteria is too heavy or too light. * Zone size is smaller than

that of the control.