Antimicrobial susceptibility test and assay bls 206

47
Antimicrobial Susceptibility Test and Assay Hoza, A.S BLS 206

Transcript of Antimicrobial susceptibility test and assay bls 206

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Antimicrobial Susceptibility Test and Assay

Hoza, A.S

BLS 206

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Aims

• 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

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Introduction

• Susceptibility test, main purposes:

– As a guide for treatment

• Sensitivity of a given micro-organism 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. Shigellae, Salmonella typhi, Mycobactrium tuberculosis)

• Continued surveillance of the susceptibility pattern of the prevalent strains (e. g. Staphylococci, Mycobactrium tuberculosis, Gram-negative bacilli)

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Introduction

• 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

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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:

– 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:

▪ at the special request of the physician/ veterinarian

▪ or when the causative organism is resistant to the first-choice drugs

▪ or when other reasons (allergy to a drug, or itsunavailability) make further testing justifiable

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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– 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

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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)

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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)

– 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

128 64 32 16 8 4 2 C1 C2

64 32 16 8 4 2 1 C1 C2

Day 1

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

Bacterial conc.= 5*105 CFU/ml

Incubate 35 oC, over night

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Broth Dilution Method

64 32 16 8 4 2 1 C1 C2

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

64 32 16

Day 2

Record visual turbidity

Subculture non-turbid tubes

to agar plates (use 0.01 ml

standard loop)

MIC = 16 mg/ml

Day 3

Determine CFU on plates:

At 16 mg/ = 700 CFU/ml >

0.1% of 5*105 CFU/ml

MBC = 32 mg/ml

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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– 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

– 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 32 ug/ml 16 ug/ml

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Agar Dilution Method

• Procedure

– 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

MIC

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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

• 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)– 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

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Disc Diffusion Method

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Disc Diffusion Method

• Procedure (cont.)

– Place the appropriate drug-

impregnated disc on the

surface of the inoculated

agar plate

– Invert the plates and

incubate them at 35 oC, o/n

(18-24 h)

– Measure the diameters of

inhibition zone in mm

Bacterial growth

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Disc Diffusion Method

• Measurement of the diameters of inhibition zone– Measure from the edge where the growth starts,

BUT there are three exceptions• 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”

– Susceptible

– Intermediate susceptible

• Low toxic antibiotics: Moderate susceptible

• High toxic antibiotics: buffer zone btw resistant and susceptible

– Resistant

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Come on, come on, it’s

either one or the other.

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Factors Affecting Size of Zone of

Inhibition

See Table

• Inoculum density

• Timing of disc application

• Temperature of incubation

• Incubation time

• Larger zones with light inoculum and vice versa

• If after application of disc, the plate is kept for longer time at room temperature, small zones may form

• Larger zones are seen with temperatures < 35 oC

• Ideal 16-18 hours; less time does not give reliable results

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Factors Affecting Size of Zone of

Inhibition

• Size of the plate

• Depth of the agar medium (4 mm)

• Proper spacing of the discs (2.5 cm)

• Smaller plates accommodate less number of discs

• Thin media yield excessively large inhibition zones and vice versa

• Avoids overlapping of zones

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Factors Affecting Size of Zone of

Inhibition

• Potency of antibiotic discs

• Composition of medium

• Acidic pH of medium

• Alkaline pH of medium

• Reading of zones

• Deterioration in contents leads to reduced size

• Affects rate of growth, diffusion of antibiotics and activity of antibiotics

• Tetracycline, novobiocin, methicillin zones are larger

• Aminoglycosides, erythromycin zones are larger

• Subjective errors in determining the clear edge

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Quality Assurance in Antibiotic Susceptibility Test

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

co-trimoxazole 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

• 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)

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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

– The strip is placed on the

surface of agar that has

been inoculated with a

lawn of test bacteria

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• E-Test

– MIC = The point (read from scale) where the zone

of inhibition intersect the strip

MIC

Antimicrobial Gradient Strip

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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)

• The lowest dilution of patient’s serum that kills bacteria

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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”– Interfere effect: reduce activity level

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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– 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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