DR.T.V.RAO MD 1

Dr.T.V.Rao MD

ANTIBIOTIC SENSITIVITY TESTING MINIMUM INHIBITORY

CONCENTRATIONSKILL BASED LEARNING

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• The responsibility of the microbiology laboratory includes not only microbial detection and isolation but also the determination of microbial susceptibility to antimicrobial agents. Many bacteria, in particular, have unpredictable susceptibilities to antimicrobial agents, and their susceptibilities can be measured in vitro to help guide the selection of the most appropriate antimicrobial agent.

WHY ANTIBIOTIC SUSCEPTIBILITY TESTING

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WHAT IS SUSCEPTIBILITY TO ANTIBIOTICS

• The term susceptible means that the microorganism is inhibited by a concentration of antimicrobial agent that can be attained in blood with the normally recommended dose of the antimicrobial agent and implies that an infection caused by this microorganism may be appropriately treated with the antimicrobial agent. The term resistant indicates that the microorganism is resistant to concentrations of the antimicrobial agent that can be attained with normal doses and implies that an infection caused by this microorganism could not be successfully treated with this antimicrobial agent.

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ANTIMICROBIAL RESISTANCE RESULTS FROM ….

Results from misuse, overuse, under/ inadequate use ofantimicrobials

• Costs money, lives and undermines effectiveness of health delivery programs

• Threat to global stability and national security

WHO Global Strategy for Containment of AntimicrobialResistance:• Intervention framework to slow emergence and reduce the spread of

antimicrobial resistant microorganisms

ANTIBIOTIC RESISTANT INFECTIONS

Diseases Agent ResistancesPneumonia S pneumoniae Penicillin

Dysentery S dysenteriae Multiple resistances

Typhoid S typhi Multiple resistances

Gonorrhea N gonorrhea Penicillin and tetracycline

Tuberculosis M tuberculosis Rifampicin and INH

Nosocomial infections S aureus Methicillin, vancomycin

E species Vancomycin

Klebsiella, Pseudomonas

Multiple resistances

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ANTIMICROBIAL SUSCEPTIBILITY TESTS

Minimum inhibitory concentration [MIC]

• The smallest concentration of antibiotic that inhibits the growth of organism

Liquid media (dilution) allows MIC estimation

Solid media (diffusion)

• Disk diffusion (Kirby-Bauer)

• E-tests

• Allows MIC estimation

Beta lactamase production: quick screening method

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SUSCEPTIBILITY TESTING: THE BIG GAPS

• MICs help, but hard to standardize

• Correlations appear possible based on individual isolates. Broad correlations based on multiple isolates are still lacking

• Understanding this helps a lot when trying to correlate outcome with MIC

- Some patients get better despite MICs

- Some patients just don’t get better despite MICs

• No rule when it correlates/not correlates

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THE TUBE DILUTION TEST IS STANDARD METHOD

• The tube dilution test is the standard method for determining levels of microbial resistance to an antimicrobial agent Serial dilutions of the test agent are made in a liquid microbial growth medium which is inoculated with a standardized number of organisms and incubated for a prescribed time. The lowest concentration (highest dilution) of test agent preventing appearance of turbidity (growth) is considered to be the minimal / minimum inhibitory concentration (MIC). At this dilution the test agent is bacteriostatic.

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• The MIC or minimum inhibitory concentration test determines antimicrobial activity of a material against a specific bacteria.

• The most commonly employed methods are the tube dilution method and agar dilution methods. Test products that are not clear or precipitate the growth media are tested by agar dilution methods which is about same as tube dilution method except dilutions are plated on agar

MINIMUM INHIBITORY CONCENTRATION TEST DETERMINES

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• The Minimum Inhibitory Concentration (MIC) is the smallest concentration of an antimicrobial agent that inhibits the growth of bacteria. The value is obtained in a highly mechanized fashion, but this procedure only provides interval censored reading. It is often of interest to use data collected from complex experiments to see how the mean MIC is affected by different factors

MINIMUM INHIBITORY CONCENTRATION (MIC)

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• The minimal bactericidal concentration (MBC) or the minimum lethal concentration (MLC) of an antibacterial which is defined as the maximum dilution of the product that will kill a test organism can be determined by sub culturing last clear MIC tube onto growth medium and examining for bacterial growth. Serial dilutions are made of the products in bacterial growth media.

THE MINIMAL BACTERICIDAL CONCENTRATION

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A MINIMUM BACTERICIDAL CONCENTRATION TEST

Figure 10.12

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MINIMUM INHIBITORY CONCENTRATION (MIC)PRACTICE THE EXERCISE FOR SKILLS

• 1. Grow cultures up overnight.2. The following day, inoculate a fresh culture with a 1:10-20 dilution and grow up to OD600 0.4. 3. Dilute to OD600 0.0005 (1:800) in THB (or relevant media).4. Dilute this 1:200 and drop about 25µl on THA (or relevant agar plates) to confirm equal starting inoculums.5. Put 50µl of the 0.0005 cultures in a 96 well plate in triplicates.6. Add 50µl per well of the substance you want to test (ie. antimicrobial peptide, antibiotic) to each well, keeping in mind the final concentration will be half the original concentration you are adding. It is good to test 1:2 dilutions (in THB, or relevant media) of this substance.7. Incubate overnight and check by eye or OD600 24 hours later to determine what concentration inhibited the growth of the different strains of bacteria.

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DILUTION IN LIQUID BROTH• Tubes containing increasing antibiotic concentrations

• Incubation during 18 hr at 37°C

• Tedious

0 (Control) 0,25 0,50 1 2 4 8 mg/l

MICBacterial growth Inhibition

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STANDARD STRAINS FOR QUALITY ASSURANCE

Precision and accuracy ensured through control strains• Known susceptibility to antimicrobial agents

Standard strains include

• Staphylococcus aureus ATCC 25923

• Escherichia coli ATCC 25922

• Pseudomonas aeruginosa ATCC 27853

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Use standardized reference

National Committee for Clinical Laboratory Standards (USA)

Other norms

• Canadian

• Chinese

• National

Do not confuse the different tables

Choose one for everything

DIFFERENT STANDARDSCLSI STANDARDS FOLLOWED ALL OVER THE

WORLD

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1. Culture media: Muller-Hinton

2. Reagents: disks

3. Size of the inoculums

4. Incubation condition

5. Control with reference strains

6. Reading inhibition diameters (accurate measurement)

7. Knowledge of staff

CRITICAL POINTS IN QUALITY ASSURANCE- ORGANISE YOUR LABORATORY WITH

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PRINCIPLES OF MINIMUM INHIBITORY CONCENTRATION

• In minimum inhibitory concentration dilute on a log2 scale each antimicrobial agent in broth to provide a range of concentrations and to inoculate each tube or, if a micro plate is used, each well containing the antimicrobial agent in broth with a standardized suspension of the microorganism to be tested. The lowest concentration of antimicrobial agent that inhibits the growth of the microorganism is the minimal inhibitory concentration (MIC). The MIC and the zone diameter of inhibition are inversely correlated . In other words, the more susceptible the microorganism is to the antimicrobial agent, the lower the MIC and the larger the zone of inhibition. Conversely, the more resistant the microorganism, the higher the MIC and the smaller the zone of inhibition.

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Minimum Inhibitory Concentration (MIC) :

Principle:

The tube dilution test is the standard method for determining levels of resistance to an antibiotic.

Serial dilutions of the antibiotic are made in a liquid medium which is inoculated with a standardized number of organisms and incubated for a prescribed time.

The lowest concentration of antibiotic preventing appearance of turbidity is considered to be the minimal inhibitory concentration (MIC).

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Different concentrations of Gentamycin in Nutrient broth:

Conc. in mcg/ml

0.1 0.2 0.4 0.8 1.6 3.1

Gentamicin, generally considered a bacteriocidal antibiotic, for this bacterium, has an MIC of 0.8 mcg/ml

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Different concentrations of Tetracycline in Nutrient broth:

Conc. in mcg/ml0.1 0.2 0.4 0.8 1.6 3.1 6.3 12.5

Tetracycline, generally considered a bacteriostatic antibiotic, for this bacterium, has an MIC of 1.6 mcg/ml

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MINIMUM INHIBITORY CONCENTRATION TEST

Figure 10.10

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The main concept is the “clinical categorisation"

• Strains are sorted according to level of Minimal Inhibitory Concentration (MIC) versus reference breakpoints

• c and C are the minor and major breakpointsSusceptible Intermediate Resistant

MIC < c ≤ MIC <

C ≤ MIC

INTERPRETATION

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UNDERSTANDING BREAKPOINTSWords of laboratory specialists

• It is not possible to work alone

• Breakpoints are the expression of a consensus among the scientific community at a given time in a country

Breakpoints are determined using two approaches• Pharmacological concept• Epidemiological concept

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MICc

Wild type

Inherited resistance

mechanism

C

THE EPIDEMIOLOGICAL CONCEPT FOR BREAKPOINTS

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THE PHARMACOLOGICAL CONCEPT FOR BREAKPOINTS

The concentration range tested for a drug and theinterpretative criteria for various categories are based onextensive studies that correlate with

• Serum achievable levels for each antimicrobial agent

• Particular resistance mechanisms

• Successful therapeutic outcome

In practice situations the entire range may not be used fordecision making and therefore the concept of breakpointconcentration

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FROM BREAKPOINTS TO INTERPRETATION

Measuring antimicrobial sensitivity of a strain isolated from a patient, to determine its status as S, I or R is an individual problem

Defining the status of a bacterial species or genus is an epidemiological problem distributed across time and space that requires monitoring

MIC ≤ c Sensitive strain

MIC > C Intermediate strain

c < MIC ≤ C Resistant strain

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Sometime the agent can still be used

• Higher doses required to ensure efficacy

• Agent may be efficacious if concentrated in vivo in an infected body fluid (e.g., urine)

Sometimes there is uncertainty • Intermediate resistance may

represent a “buffer” zone that prevents strains with borderline susceptibility from being incorrectly categorized as resistant

INTERPRETING INTERMEDIATE RESISTANCE

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COMMON INTERPRETATION PROBLEMS

Results depends on the technique used

Many factors influence results

• Lack of standardization of the inoculums

• Thickness and quality of the culture media

• Quality and conservation of the disks

• Quality control with standardized strains

• Condition and duration of incubation

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• E-test is based on arraying a concentration gradient of each antibiotic on a polymer strip. Concentration values are marked on the other side of the strip so that one can easily locate corresponding concentrations. E-strips, also known as “epsilometers”, are commercially prepared by micro dispersing robotic machines that can deliver Nano liter volumes of antibiotic concentration along the strip.

WHAT IS E-TEST

E-TESTPlastic strips with a predefined gradient of

• One antibiotic

• One antifungal

Only one manufacturer

One strip per antibiotic

Wide range of antibiotics

Easy to use

Storage at -20°C

Short shelf life, expensive

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AN E-TEST COMBINES ASPECTS OF KIRBY-BAUER AND MIC TESTS

Figure 10.11

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READING E-TESTS

Susceptible < 1

Resistant > 4 ug/ml

Ciprofloxacin for Yersinia pestis

Intermediate 1-4 ug/ml

Upper reading

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Problems with E-test reading

COMMON INTERPRETATION PROBLEMS

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• The Liofilchem MIC Test Strip is a quantitative assay for determining the Minimum Inhibitory Concentration (MIC) of antimicrobial agents against microorganisms to indicate appropriate patient treatment and for identifying resistance patterns

NEW LIOFILCHEM MIC TEST STRIPS

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• When the Liofilchem MIC Test Strip is applied onto an inoculated agar surface, the preformed exponential gradient of antimicrobial agent is transferred into the agar matrix. After 18 hours incubation or longer, a symmetrical inhibition ellipse centered along the strip is formed. The MIC is read directly from the scale in terms of µg/mL, at the point where the edge of the inhibition ellipse intersects with the MIC Test Strip.

NEW LIOFILCHEM MIC TEST STRIPS

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• Oxoid has l improved M.I.C.Evaluator™ (M.I.C.E.™) strips, a product range for the accurate determination of minimum inhibitory concentration (MIC) values. The new distinctive gradient format of M.I.C.E. strips provides an excellent contrast with agar and the increased font size makes reading easier.

M.I.C.EVALUATORS (M.I.C.E.) SIMPLE, CONVENIENT METHOD FOR ACCURATE

MIC VALUES

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ROLE OF NCDC ( INDIA ) IN CONTAINMENT OF ANTIMICROBIAL RESISTANCE (AMR)

• Antimicrobial resistance in pathogens causing important communicable diseases has become a matter of great public health concern globally including our country. Resistance has emerged even to newer, more potent antimicrobial agents like carbapenems. The factors responsible for this are widespread use and availability of practically all the antimicrobials across the counter meant for human, animal and industrial consumption. There are definite policies /guidelines for appropriate use of antimicrobials at national level in specific national health programme being run in the country

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IMPLEMENTATION OF WHONET CAN HELP TO MONITOR RESISTANCE

• Legacy computer systems, quality improvement teams, and strategies for optimizing antibiotic use have the potential to stabilize resistance and reduce costs by encouraging heterogeneous prescribing patterns and use of local susceptibility patterns to inform empiric treatment.

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ANTIBIOTIC RESISTANCE A CONCERN TO

HUMANITY ???

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• Created by Dr.T.V.Rao MD for ‘ e ‘ learning resources for Medical Microbiologists in the

Developing World

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