Antimicrobial Susceptibility Test and AssayHoza, A.SBLS 206

Aims

• be able to describe:– The methods of antimicrobial susceptibility testing

– Factors affecting antimicrobial activity

– Quality assurance of antibiotic susceptibility testing

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

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)

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

IntroductionAntimicrobial agents commonly used to treat

systemic infection

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)

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-ch

oice drugs▪ or when other reasons (allergy to a drug, or its unavailabi

lity) make further testing justifiable

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

Set 1 Set 2

Staphylococcus Benzyl penicillinOxacillinErythromycinTetracyclineChloramphenicol

GentamicinAmikacinCo-trimoxazoleClindamycin

Intestinal AmpicillinChloramphenicolCo-trimoxazoleNalidixic acidTetracycline

Norfloxacin

Enterobacteriaceae

Urinary

SulfonamideTrimethoprimCo-trimoxazoleAmpicillinNitrofurantoinNalidixic acidTetracycline

NorfloxacinChloramphenicolGentamicin

Blood and tissues AmpicillinChloramphenicolCotrimoxazoleTetracyclineGentamicin

CefuroximeCeftriaxoneCiprofloxacinPiperacillinAmikacin

Pseudomonas aeruginosa PiperacillinGentamicinTobramycin

Amikacin

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

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)

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

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

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 bacteriaBacterial conc.= 5*105 CFU/ml

Incubate 35 oC, over night

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 3Determine CFU on plates:At 16 mg/ = 700 CFU/ml > 0.1% of 5*105 CFU/ml

MBC = 32 mg/ml

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

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

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

Microbroth Dilution Method

• Visualize turbidity– Light box/ mirror reader– Automated reader

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

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

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

Disc Diffusion Method

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 st

andard.* – 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

Disc Diffusion Method

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

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

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

Come on, come on, it’s either one or the other.

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

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

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

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

Quality Assurance in Antibiotic Susceptibility Test

• Media recommended for test of fastidious bacteria

Quality Assurance in Antibiotic Susceptibility Test

• Media recommended for test of fastidious bacteria

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)

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 sensitivit

y determination– Use appropriate control cultures– Use standard methodology for the test

Quality Assurance in Antibiotic Susceptibility Test

• Salient features of quality control – Use coded strains from time to time for internal qua

lity control– Keep the antibiotic discs at room temperature for o

ne hour before use– Incubate the sensitivity plates for 16-18 hours befor

e reporting– Incubate the sensitivity plates at 35oC– Space the antibiotic discs properly to avoid overlap

ping of inhibition zone

Quality Assurance in Antibiotic Susceptibility Test

• Salient features of quality control – Use inoculum size that produces ‘near confluent’ g

rowth– Ensure even contact of the antibiotic disc with the

inoculated medium– Measure zone sizes precisely– Interpret zone sizes by referring to standard chart

s

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

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

• E-Test– MIC = The point (read from scale) where the zone

of inhibition intersect the strip

MIC

Antimicrobial Gradient Strip

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

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

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

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