Cmb508302_ab Susceptibility Test_2548

Antimicrobial Susceptibility Test and Assay Usanee Anukool (Ph.D.) Clinical Microbiology, AMS, Chiang Mai University 5 Jan 2006

Aims  After class, students will be able to describe: 

The methods of antimicrobial susceptibility testing



Factors affecting antimicrobial activity



Quality assurance of antibiotic susceptibility testing

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Contents  Introduction  Antimicrobial Susceptibility Test and Assay

Dilution methods  Disc diffusion method  Factors affecting size of zone of inhibition 



 Quality Assurance in Antibiotic Susceptibility

Testing 5-Jan-06

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Introduction  Susceptibility test, main purposes:  As a guide for treatment  

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As an epidemiological tool 



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Sensitivity of a given m.o. to known conc. of drugs Its concentration in body fluids or tissues

The emergence of resistant strains of major pathogens (e. g. Shigellae, Salmonella typhi) Continued surveillance of the susceptibility pattern of the prevalent strains (e. g. Staphylococci, Gram-negative bacilli)

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Introduction  Methods for antimicrobial susceptibility testing 

Indirect method 



Direct method  

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cultured plate from pure culture

Pathological specimen e.g. urine, a positive blood culture, or a swab of pus

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Introduction  Antimicrobial agents commonly used to treat systemic

infection

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Introduction  Inoculum preparation

- Number of test organisms can be determined using different methods:    

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Direct count (Microscopic examination) The optical density (OD) at 600 nm (Spectrophotometry) Plate count: making dilution first Turbidity standard (McFarland)

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Introduction  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:  



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at the special request of the physician or when the causative organism is resistant to the firstchoice drugs or when other reasons (allergy to a drug, or its unavailability) make further testing justified

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Table 1: Basic sets of drugs for routine susceptibility tests (http://w3.whosea.org/) Set 1

Set 2

Staphylococcus

Benzyl penicillin Oxacillin Erythromycin Tetracycline Chloramphenicol

Gentamicin Amikacin Co-trimoxazole Clindamycin

Intestinal

Ampicillin Chloramphenicol Co-trimoxazole Nalidixic acid Tetracycline

Norfloxacin

Enterobacteriaceae Urinary

Sulfonamide Trimethoprim Co-trimoxazole Ampicillin Nitrofurantoin Nalidixic acid Tetracycline

Norfloxacin Chloramphenicol Gentamicin

Blood and tissues

Ampicillin Chloramphenicol Cotrimoxazole Tetracycline Gentamicin

Cefuroxime Ceftriaxone Ciprofloxacin Piperacillin Amikacin

Pseudomonas aeruginosa

Piperacillin Gentamicin Tobramycin

Amikacin

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Antimicrobial Susceptibility Testing  Dilution method

vary amount of antimicrobial substances incorporated into liquid or solid media  followed by inoculation of test bacteria 

 Diffusion method 

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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 Chiang Mai University

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Dilution Method  Broth dilution/ Agar dilution methods  Permit quantitative results: 

Indicating amount of a given drug necessary to inhibit (bacteriostatic activity) or kill (bactericidal activity) the microorganisms tested

 Minimum Inhibition Concentration (MIC)  Minimum Bactericidal Concentration (MBC) 5-Jan-06

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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) 

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The lowest concentration of antimicrobial agent that allows less than 0.1% of the original inoculum to survive

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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  Subculturing of non-turbid tubes, overnight  Growth (bacterial count)  MBC 

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Broth Dilution Method Day 1 128 64

32

16

8

4

2 C1 C2

64

16

8

4

2

1 C1 C2

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Bacterial conc.= 5*105 CFU/ml Incubate 35 oC, o/n 5-Jan-06

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

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Broth Dilution Method Day 2 64

32

16

8

4

2

1 C1 C2

Record visual turbidity Subculture non-turbid tubes to agar plates (use 0.01 ml standard loop)

0.01 ml (spread plate), Incubate 35 oC, o/n

64

32

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MIC = 16 mg/l Day 3 Determine 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 plate subcultured with 0.01 ml 

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500*0.01 = 5 CFU

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Broth Dilution Method  Disadvantages :

Only one antibiotic & one organism can be tested each time  Time-consuming 

 Solutions??

Agar dilution method  Disc diffusion method  Microbroth dilution method 

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Microbroth Dilution Method  Microdilution plates:  

“Microdilution/ Microbroth dilutions” 96 wells/ plate: simultaneously performed with many tests organisms/ specimens, less reagent required

 Manually prepared  Commercially prepared   

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Frozen or Dried/ lyophilized Consistent performance but high cost May suffer from degradation of antibiotic during shipping and storage

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Microbroth Dilution Method  Visualize turbidity  Light box/ mirror reader  Automated reader

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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 ug/ml 5-Jan-06

32 ug/ml Chiang Mai University

16 ug/ml 20

Agar Dilution Method  Procedure 

Inoculation of bacterial inoculum (McFarland No. 0.5) 



Using a replicating inoculator device called “A SteersFoltz replicator” Delivers 0.001 ml of bacterial inoculum

Incubation  Spot of growth 

MIC 5-Jan-06

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32 ug/ml

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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 

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Demonstrated that the qualitative results of filter disk diffusion assay correlated well with quantitative results from MIC tests

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Disc Diffusion Method

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Disc Diffusion Method  Procedure (Modified Kirby-Bauer method: National Committee for Clinical Laboratory Standards. NCCLS) 



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Prepare applx. 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 Chiang Mai University

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Disc Diffusion Method

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Disc Diffusion Method  Procedure (cont.) 



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Place the appropriate drugimpregnated disc on the surface of the inoculated agar plate Invert the plates and incubate them at 35 oC, o/n (18-24 h)

Bacterial growth

Measure the diameters of inhibition zone in mm

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Disc Diffusion Method  Measurement of the diameters of inhibition zone 

Measure from the edge where the growth stats, BUT there are three exceptions 

 

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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

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Disc Diffusion Method  Interpretation of results 

By comparing with the diameters with “standard tables”

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Susceptible Intermediate susceptible

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 

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Low toxic antibiotics: Moderate susceptible High toxic antibiotics: buffer zone btw resistant and susceptible

Resistant

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Factors Affecting Size of Zone of Inhibition See Table 2.  Inoculum density

 Timing of disc

application

 Larger zones with light

inoculum and vice versa

 If after application of disc, the

plate is kept for longer time at r oom temperature, small zones may form

 Temperature of

 Larger zones are seen with

 Incubation time

 Ideal 16-18 hours; less time

incubation

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temperatures < 35 oC

does not give reliable results

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Factors Affecting Size of Zone of Inhibition  Size of the plate

 Smaller plates accommodate

 Depth of the agar

 Thin media yield excessively

 Proper spacing of

 Avoids overlapping of zones

medium (4 mm)

less number of discs

large inhibition zones and vice versa

the discs (2.5 cm)

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Factors Affecting Size of Zone of Inhibition  Potency of antibiotic

 Deterioration in contents leads to

 Composition of

 Affects rate of growth, diffusion of

discs

medium

 Acidic pH of medium

reduced size

antibiotics and activity of antibiotic s

 Tetracycline, novobiocin,

methicillin zones are larger

 Aminoglycosides, erythromycin

 Alkaline pH of

medium

 Reading of zones

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zones are larger

 Subjective errors in determining

the clear edge

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Quality Assurance in Antibiotic Susceptibility Test  WHO-Regional Office for South East Asia website  Medium: Mueller-Hinton agar plates 

Enterococcus faecalis (ATCC 29212 or 33l86) and a disc of cotrimoxazole 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 

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Quality Assurance in Antibiotic Susceptibility Test  Media recommended for test of fastidious bacteria

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Quality Assurance in Antibiotic Susceptibility Test  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 27853 

 See Table 3.

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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 

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Quality Assurance in Antibiotic Susceptibility Test  Salient features of quality control

Use coded strains from time to time for internal quality control  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 

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Quality Assurance in Antibiotic Susceptibility Test  Salient features of quality control

Use inoculum size that produces ‘near confluent’ growth  Ensure even contact of the antibiotic disc with the inoculated medium  Measure zone sizes precisely  Interpret zone sizes by referring to standard charts 

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Quality Assurance in Antibiotic Susceptibility Test  Frequency of quality control test (Fig 1.)

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Antimicrobial Gradient Strip  E-Test  Antibiotic was applied to one side  Interpretive scale printed on another side 

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The strip is placed on the surface of agar that has been inoculated with a lawn of test bacteria

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Antimicrobial Gradient Strip  E-Test  MIC = The point (read from scale) where the zone of inhibition intersect the strip

MIC

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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) 

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The lowest dilution of patient’s serum that kills bacteria Chiang Mai University

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Activity of Combined Drugs  The combination of drugs used when:  Serious infection  Organisms with high rate of resistance 



E.g. Mycobacterium tuberculosis

In immunosuppressive patients

 “Synergistic”  Additive effect: increase in activity level  “Antagonistic”  5-Jan-06

Interfere effect: reduce activity level Chiang Mai University

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Activity of Combined Drugs  “Synergistic”  E.g. aminoglycosides and penicillins  “Antagonistic”  e. g. Penicillins and bacteriostatic drugs such as tetracyclines are antagonistic, since penicillins require actively growing cells

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Antibiotic resistant bacteria  Nosocomial infection / Hospital-acquired รืดำำ

แรนื  



ESBL (Extended beta-lactamase) MRSA (Methicillin resistant Staphylococcus aureus) Oxacillin PRSP (Penicillin resistant Streptococcus pneumoniae)  Oxacillin •Combined drug assay

•Amoxicillin/ Clavulanic acid (AMC) •ESBL producing strain

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References  Brooks, G.F., J.S. Butel, S. A. Morse. 1998. Jawetz,

Melnick & Adelberg’s Medical Microbiology, 21st ed., Prentice Hall International Inc. USA.  http://w3.whosea.org/en/section10/section17/ section53/section482_1787.htm  http://w3.whosea.org/en/Section10/Section17/ Section53/Section375_1185.htm  National Committee For Clinical Laboratory Standards. 1998. NCCLS document M100 - S8 . Perf ormance Standards for Antimicrobial Susceptibility Te sting. 8th edition, NCCLS, Waynae, Pa.

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