UJI SENSITIVITAS BAKTERI

Learning objectives

A. Mechanism of antibiotic resistant

B. Understand standard antimicrobial susceptibility testing

C . Interpret antimicrobial susceptibility testing

Antimicrobial resistant: Results f r o m
misuse, overuse, u n d e r / inadequate use of
antimicrobials
❖ Costs money, lives and undermines
effectiveness of health delivery
programs

❖ Threat to global stability and national

security
 Susceptibility test, main purposes

AS a guide for treatment

• Antim icrobial sensitivity testing : to determine the most suitable

ant ibiot i c agent again st specific m icroorganism (d r ug of choice)
.WHY?

• W idespread and non selective a n tibiot ics usage r esults in a high

driving force in the development of antibiotic resistances

As an epidemiological tool
• The emergence of resistant strains of m ajor pathogens (e. g.
Shigellae, Salmonella typhi, Mycobacterium tuberculosis)
• Continued surveillance of the susceptibility pattern of the
prevalent strains (e. g. Staphylococci, Mycobacterium tuberculosis,
Gram-negative bacilli)
 Direct versus indirect susceptibility tests

Indirect method
• cultured plate f r o m pure culture .
Colony pick up adjust turbidity spread the inoculum

Direct method
•Pathological specimen • e.g. urine, a positive blood culture, or a
swab of pus
 Common methods used for AST

1. Agar diffusion based methods (qualitative method)

• Disc diffusion method (also called Kirby-Bauer method)
• Epsilometer test (E-test)
2.Dilution method (Quantitative method) :Rare used
Principle

The Kirby-Bauer ( K - B ) is the most common method for antibiotic

resistance/susceptibility testing. The results of such testing help
physicians in choosing which antibiotics to use when treating a sick
patient

Test utilizes small filter disks impregnated wi t h a known

concentration of antibiotic. The disks are placed on a Mueller-tfinton
agar plate t h a t is inoculated w i t h the test microorganism.

Upon incubation, antibiotic diffuses f r o m the disk into the

surrounding agar. If susceptible to the antibiotic, the test organism
will be unable t o grow in the area immediately surrounding the disk,
displaying a zone of inhibition .
Mueller-tfinton agar (MtfA)

M t f A is the best m e d ium to use for routine susceptibility testing

using Kirby-Bauer disc diffusion method for nonfastidious bacteria
(both aerobe and facultative anaerobe). Use of media other than
Mueller-tfinton agar m ay result in erroneous results.
Formula for Mueller-Hinton agar per liter of purified water
• Beef extract: 2 . 0 g
• Acid tfydrolysate of Casein: 1 7 . 5 g
• Starch: 1 . 5 g
• Agar: 1 7 . 0 g

Placing antibiotic disc in Mueller-Hinton

agar medium
Why mueller-hinton agar is used in routine
antibiotic susceptibility testing (AST) ?

• I t shows acceptable batch - to- batch reproducibility for

susceptibility testing
• I t supports satisfactory growth of most nonfastidious pathogens
• I t is low in sulfonamide, t r i m e t h o pr i m , and tetracycline inhibitors
(i.e. concentration of inhibitors thymidine and thym ine is low in
Mtf A)
• A large body of data and experience has been collected
concerning susceptibility tests performed wi t h this medium.
 Modifications of Muller Hinton agar
• Mueller Hinton agar medium supplemented with 5% sheep
blood is recommended for determining the antimicrobial
susceptibility of
– Streptococcus pneumoniae
– Neisseria meningitidis

The disk diffusion test. Virulent Streptococcus pneumoniae on the surface

of Mueller - t finton agar w i t h 5% horse blood. Susceptible t o all tested
antibiotics.
Procedure of K - B methods

1. Clinical specimens e.g urine

2. Isolation and identification e.g E.coli
• Note bacterial infections are usually caused by one pathogenic
bacteria except anaerobic infections which are mixed infection
3.Preparation of inoculum
3. Preparation of inoculum
• Using a sterile inoculating loop, touch four or five isolated colonies
of the organism to be tested.
• Suspend the organism in 2 m l of sterile saline.
• Vortex the saline tube to create a smooth suspension.
• Adjust the turbidity of this suspension to a 0 . 5 McFarland
standard by adding more organism if the suspension is too light.
• Use this suspension wi t h i n 1 5 minutes of preparation.
 McFarland Nephelometer Standards

In microbiology, McFarland standards are used as a reference to adjust the

turbidity of bacterial suspensions, if the bacterial suspension is too turbid,
it can be diluted with more diluent. If the suspension is not turbid enough,
more bacteria can be added
4. Dip a sterile cotton swab into bacterial suspension, after mixing
i t then remove the swab and excess fluid is expressed by pressing
and rotating the swab against the inner sides of the tube above
the level of the liquid.
5 - S t r e a k the swab all over the surface of the sensitivity testing
m edium (Mueller-tf inton agar) three times in different directions,
rotating the plate through an angle of 6 0 º after each application .
Finally, pass the swab around the edge of the agar surface, by this
you get a lawn growth. Discard the swab in disinfectant, leave the
plate to d r y a few minutes a t room temperature wi t h the lid closed.
(the depth of the m e d ium in Petri dish 4 m m → 2 5 m l of the
medium).
6 - P i c k up an antimicrobial disc wi t h the sterile forceps and place i t on the
surface of the agar medium, each disc should be gently pressed down to
ensure even contact wi t h the medium, using the tips of the forceps. Six
discs can be placed on 9 0 m m plate .five discs m ay be spaced evenly about
1 5 m m f r o m the edge of the plate and one disc in the center . Also can be
used antibiotic disc dispenser to apply discs on surface of the medium.
7 - Invert the plates and incubate a t 37Cº for 1 8 to 2 4 hours.
A f t e r incubation the diameter of each inhibition zone (including the
diameter of the disc) should be measured and recorded in m m . the
measurements can be made wi t h the ruler f r o m the back (under
surface) of the plate wi t h out opening the lid. The diameter of each
inhibition zone is compared to the standard charts .
Inhibition zone:-
The area where no bacteria grow
after overnight incubation
 Interpretation and Reporting of the Results

• Using the published EUCAST guidelines, determine the

susceptibility or resistance of the organism to each drug tested
(Table 1).
• For each drug, indicate on the recording sheet whether the zone
size is susceptible (S), intermediate (I), or resistant (R) based on
the interpretation chart.
• The results of the Kirby-Bauer disk diffusion susceptibility test are
reported only as susceptible, intermediate, or resistant. Zone
sizes are not reported to physicians.
 Depend on the diameter of inhibition zone ,the bacteria
divided to : -

Sensitive S →it means the bacterium responds t o the

antimicrobial agent.

Intermediate I →it means the bacterium is n o t fully

sensitive t o the antimicrobial agent.

Resistant R →it means the bacterium is n o t effected by the

antimicrobial agent.
What factors influence the size of the zone
of inhibition ?

1. Pathogen susceptibility
2. Antibiotic diffusion effects
3. Agar depth : 4 m m is good ,to avoid false susceptible or resistant
4. ptf: 7 . 2 to 7 . 4 p t f
5. Size of the inoculated organism:
6. Presence of other metals :Excessive thym idine or thym ine can
reverse the inhibitory effects of sulfonamides and t r i m e t h o p r i m
resulting in smaller and fewer distinct zones of inhibition, or no
zones a t all.
7. the temperature and length of incubation

Microorganisms t h a t are resistant to an antibiotic will not show a

zone of inhibition (growing right up to the disk itself) or display a
relatively small zone.
E- TEST

The E test (also known as the Gradinet Diffusion Method) is based

on the same principle as the disk diffusion method.

I t is an in vitro method for quantitative antimicrobial susceptibility

testing

E-Test determines the M i n i m u m Inhibitory Concentration (MIC).

Following incubation, the MIC value (ug/m l) is read a t the point of
intersection between the zone edge and the E-Test strip.
Materials
• E-test strips (store frozen)
• Mueller tf inton Agar (Mtf)

Procedures
• The same suspension of the test organism in sterile saline
equivalent to a 0 . 5 McFarland standard using isolated colonies
will be used as in the previous experiment (disk method).
• Using a sterile cotton swab, inoculate the organism onto an
appropriate agar plate, streaking in 3 directions over the entire
agar surface.
• Using needle, apply the Imipenem antimicrobial strip onto the
agar.
• Incubate plate a t 35 o C x 2 0 to 2 4 hours
Interpretation OF E.Test

• A f t e r incubation read the MIC value a t the point of intersection

between the zone edge and the E- te s t strip. Since E- test
comprises a continuous gradient, MIC values in between t wo - f o l d
dilutions can be obtained. Always round up these values to the
next t wo - f o l d dilution before interpretation.
Dilution tests (Quantitative):
This test is used t o determine the M i n i m u m Inhibitory Concentration
(MIC) of antimicrobial agent and M i n i m u m Bactericidal
Concentration(MBC)
e,g. Broth (Tube) dilution m ethod : -
This test can be done by adding the same volume of standardized
inoculum to a series of tubes containing t wo fold dilution in nutrient
broth of the antimicrobial being test. A f t e r overnight incubation a t
37Cº the tube t h a t shows no growth (no turbidity) wi t h lowest
concentration of drug represents MIC. Also MBC can be determined
by inoculating loopfuls f r o m the tubes not showing growth into
suitable agar m edium .
The plate showing no growth represents MBC.

MIC:-(Minimum Inhibitory Concentration)

The lowest concentration of antimicrobial agent t h a t inhibits
bacterial growth after overnight incubation (no turbidity) .

MBC :-(Minimum Bactericidal Concentration)

The lowest concentration of antimicrobial agent t h a t kills or prevents
bacterial growth after subculture of bacteria to a media.
Any Questions?

Thank You