Antimicrobial susceptibility testing – disk diffusion methods

ANTIMICROBIAL SUSCEPTIBILITY TESTING –DISK DIFFUSION METHODS

INTRODUCTIONAntimicrobial Susceptibility Test is very important

for treating infectious diseases and monitoring antimicrobial resistance in various pathogens.

It is essential that the reports are

relevant,

timely

interpreted correctly

to ensure Quality Control.

To guide the clinician- selection of antibiotics

To accumulate epidemiological information on the resistance of microorganisms of public health

importance within the community.

DEFINITION

AST :

It is a determination of least amount of an antimicrobial chemotherapeutic agent that will inhibit the growth of microorganism invitro.

Quality control :

A process in the laboratory designed to monitor the analytical phase of testing procedure to ensure that tests are working properly.

AST methods

a. Disk diffusion method:

1. Kirby Bauer method

2. Stokes method

b. MIC:

1. Broth dilution method

2. Agar dilution method

c. E-test

Diffusion-Kirby Bauer method

Principle

Paper disks impregnated with antimicrobial agent are placed on agar medium uniformly seeded with the test organism.

A concentration gradient of the antibiotic is formed by diffusion from the disk and the growth of the test organism is inhibited at a distance form the disk (that is related among other factor) to the susceptibility of the organism

Medium

According to CLSI (clinical laboratory standard institute)

Muller Hinton Agar - Non fastidious organism

Temperature - 45°C to 50°C

Thickness 4mm

PH 7.2 – 7.4

Moisture

Storage: 5 days at 2-8°C

Prolonged storage causes – dehydration of the media

MHA Plates wrapped in air tight plastic bags and refrigerated – 2 weeks

Media used

Muller Hinton Agar

It is best for non fastidious organism

It shows acceptable batch to batch reproducibility

It has low thymidine content.

(Increased thymidine antagonise the activity of sulphonamides)

Reverse the inhibitory effect of SXT – lesser or no zone-falls resistant report

To check QC – ATCC 29212

E.faecalis – SXT ->20mm

MHBA ( 5% sheep blood agar )

Strept. Pneumoniae

Beta strept, alpha strept, non haemolytic strept

MHCA & HTM

Haemophilus spp

GC agar

Gonococci

Media used contd

Antibiotics

Commercial diskWhenever we receive the antibiotics check the label,

Mfg date, Exp date and Lot no. It should be checked with ATCC strainsStored at -20°C or -70°C and at 4°C – 8°CRoutine use keep at 4°C – 8°C

Paper disk (In-house)Whatmann filter paper No. 2 is usedDiameter 6mm with regular edgesSterilize by hot air oven at 160°C for 1hourDo not use irregular edged and charred disk

Antibiotic solution preparation

It is always prepared from pure substance

Stock made concentrations depending on disk strength

Some antibiotics dissolved in organic solvent and others in sterile distilled water

Use only minimum volume of organic solvent to stabilize the antimicrobial powder

After preparing the solution should checked with ATCC strains

Prepared antibiotics are aliquote into 6-7 ml in tubes

Lesser amount – improper delivery of antibiotic

Antibiotic solution preparation

Eg; Ampicillin – Needed concentration-2000µg/ml(DD strength 10µg/ml)

1mg=1000µg/ml

2mg=2000µg/ml

20mg=2000µg/10ml

200mg=2000µg/100ml

Volume stock (in ml) =weight(mg) x potency of antibiotic(µg) /needed concentration(µg)

Obtaining satisfactory results, dispense 10ml into 20ml sterile tube

Store it at -20°C for six months

Inoculum

Turbidity standard for inoculum preparation

McFarland Standard – BaSO4

0.5 - 2 x 108 - for GNB and fast growing organism

1.0 - 3 x 108 - for Gram positive cocci

PREPARATION OF CULTURE

Select 10 morphologically identical well isolated colonies

Inoculate in 1.5ml NB

Incubate for 2hrs

Adjust opacity – McFarland's standard

0.5 for Gram Negative bacilli

1 for Gram Positive cocci

INOCULATION Marking the plates Six antibiotics – 85 to 90mm petridishes Two antibiotics – QC Streaking the plates

( within 15 mins after opacity adjusted ) Placing the disks

In-between two disk – 24mm Periphery to the disk – 15mmOverlapping of zone of inhibition should be

avoided Time duration – 15minsLoop 2mm diameter it delivers 0.005ml Ensure complete contact to the agar surfacedisk should not be relocated

INCUBATION & ATMOSPHERE

MHA plates are incubated at 37°C for 16-18hrs

MHBA, HTM are incubated at 37°C with 5% CO2 incubator

ATCC control strains for AST

QUALITY CONTROL

To check the quality of the medium

Potency of the antibiotic

Technical error

When ever we receive new drug or media

Quality control (QC)

QC - A procedure which ensures that the performance of a test/procedure is reliable

QC in AST - Testing a standard strain of known susceptibility to the antimicrobial agent tested

Goal of QC - Accuracy and reproducibility

ATCC control strains for AST Eg: ATCC 25923 Staph aureus (beta lactamase negative,

oxacillin - susceptible)

ATCC 27853 Pseudomonas aeruginosa (for aminoglycosides)

ATCC 25922 E.coli (beta lactamase negative)

ATCC 35218 E.coli (beta lactamase positive)

ATCC 29212 E.faecalis (for checking of Thymidine level of MHA)

ATCC 700603 Kleb. Pneumoniae (ESBL-positive)

ATCC 49619 Strept. Pneumoniae (oxa – R)

ATCC 49247 H.influenzae

ATCC 49766 H.influenzae

Procedure for performing QC

READING

Each zone size is interpreted according to the organism by reference in the CLSI guidelines

RESISTANCE :resistant , to indicate that the bacteria can not be inhibited by the antibiotics.

INTERMEDIATE : intermediate , to indicate that the bacteria can be inhibited by the high dose of antibiotics.

SUSCEPTIBLE :susceptible, to indicate that the bacteria can be inhibited by the normal dose of antibiotics

READING AND INTERPRETATION

Only pure growth is considered for reading

Inoculum should be adequate

There should not be any misplacing of antibiotics

Quality control strains should be in expected ranges (guided by CLSI)

Acidic p H of medium

Alkaline p H of medium

Addition of thymidine to medium

Low content of thymidine

Less action aminoglycoside, quinolones and macrolides,

excess activity of tetra

More activity of Aminoglycosides, quinolones and macrolides

Lesser activity of tetra

Decrease activity of SXT –resistant zone

ATCC 29212 E.faecalis – SXT more than – 20mm satisfactory

Magnesium + Calcium (cation)

Excess : reduce zone size for aminoglycosideLow : increase zone size for aminoglycoside

Zinc

Excess : reduce zone size for carbapenems

Lesser : increase zone size in carbapenems

Larger zone of inhibition

Light inoculumError in inoculum preparationDepth of the medium is thinMHA is nutritionally unacceptable

Smaller zone of inhibition

heavy inoculumError in inoculum preparationDepth of the medium is thick

One or more zone too small or too large zone

Measurement errorTranscription errorRandom defective diskDisk not pressed firmly to the agar surface

One QC strain is out of range but other QC strain are within range for the same antibiotic

One may be the better indicator of QC problem

Two QC strain is out of for the same antibiotic

Problem with the disk

Cont..

Reading……

Zone of inhibition measured in diameter or radius with transparent ruler

Cont…

Colonies within the zones of inhibition

Zones overlap

Zones indistinct

Mixed culture

Resistant mutants within the zone.

disks too close together

Poorly streaked plate

• Cont….

Double zone

Proteus swarming – ignore

Fastidious organism – eg, beta Strept, S.pneumoniae – zone of inhibition

not by haemolysis

Co-trimoxazole reading

PrecautionsAmpicillin is always R to Klebsiella and Aeromonas spp

Nitrofurantoin S to E.coli R to Proteus and Klebsiella

Cefoxitin R – MRSA

Cefpodoxime R – ESBL in GNBImipenem and meropenem R – CRO

Cont….

Vancomycin and teicoplanin resistant – VRE

Alert forms – HICC, MS office, respective units/wards

S.typhi and S. paratyphi A newer guideline for ciprofloxacin – >31 is S and MIC by E.test

Oxacillin R S.pneumoniae do penicillin MIC

ADVANTAGES

Technically simple to perform

Reproducible reagents are inexpensive

Does not require any special equipments

Easily understood by clinicians

Flexible regarding the selection of antibiotics

STOKES METHOD

Procedure

Stokes method

Susceptible – zone size of the test strain is larger than or equal to control strain

Resistant – zone size of the test strain is smaller than 2mm

Intermediate – zone size of the test strain is 2-3mm smaller than that of the control strain

AdvantagesBoth control and test organism is same environment

Disadvantages 2 to 4 antibiotics in one plate is tested

Laborious

Things need to

be known

Organisms requiring special considerations

• Emergence of resistance:

• Staphylococci:

• Methicillin resistant S.aureus:

• Oxacillin and other penicilinase resistant penicillin such as methicillin, cloxacillin constitute the drug of choice for Staphylococcal infections.

• Methicillin is no longer the agent of choice for testing and treatment.

• The penicillin binding protein which has low affinity for binding all beta-lactam drugs is encoded by the gene

mec A

Contd....

• mec A is responsible for resistance to methicillinand other beta lactam antibiotics

• mec A encodes penicillin binding proteins 2a, which differs from other penicillin binding protein as its active site does not bind methicillin or other beta lactam antibiotics

• Penicillin binding protein 2a can continue to catalyze the transpeptidation reaction required for peptidoglycan, enabling the cell wall synthesis in the presence of antibiotics

Contd....• Consequence of the inability of PBP2a to interact

with beta lactams

• Acquition of mecA confers resistance to all beta lactam antibiotics in addition to methicillin

• MRSA is significant in hospital acquired and community associated infections

• Drug of choice – vancomycin and teicoplanin –injectable

• Rifampacin and linezolid – oral drug

• Topical application – bacitracin, chlorohexidine, mupirocin

Detection methods for MRSA

• Cefoxitin DD – surrogate marker

• Because cefoxitin serves to induce greater expression of PBP2a in mec A containing strains of Staphylococci and also function as test reagent to detect resistant.

• Oxacillin screen plate– MHA with 4%Nacl + 6mg per ml

– Spot inoculate – incubate at 350C

– More than one colony indicates oxacillin resistant

Molecular detection by PCR can be performed

• CLSI recommends cefoxitin for specific break points interpretative criteria for S.aureus and Coagulase neg Staph.

• Cefoxitin – zone of inhibition can be easily read than oxacillin DD.

Break points for DD interpretation

Cefoxitin Oxacillin

Resistance Susceptible Resistance Intermediate Susceptible

S.aureus 21 22 10 11-12 13

CONS 24 25 17 - 18

Cefoxitin vs Oxacillin

• Cefoxitin

• Stable drug

• Requires 16-18hrs incubation at 37 0C

• No supplement is necessary

• Clear zone of inhibition and wider range of interpretative criteria

• Oxacillin

• Degradation on storage

• Requires 24hrs incubation at 350 C

• 2-5% Nacl is added

• Narrow range of interpretative criteria hence zone of inhibition is measured using transmitted light

Vancomycin resistance or diminished susceptibility in S.aureus

• Strains with reduce susceptibility to vancomycin have been called vanco intermediate S.aureus (VISA) or glycopeptideintermediate (GISA)

• Between 2002-2005 five different strain of MRSA were detected for the first time with vancomycin resistant.

• The first MRSA isolate with more subtle diminished susceptibility to vancomycin with MIC value 8mg per ml (intermediate)

• Although still uncommon both vanco(R) S.aureus and VISA are of great concern because vanco is the drug of choice for MRSA

DETECTION METHODS

• Vancomycin DD

• Vancomycin MIC

• Vancomycin agar screen test

– Brain heart infusion agar with vancomycin 6mg per ml

Inducible clindamycin resistant in Staphylococci:

• Two different resistant mechanisms confers macrolide resistance (e.g. erythromycin)

• The erm gene codes for methylation of 23S r RNA which results in resistant to erythromycin and either inducible or constitutive resistant to clindamycin.

• The msrA gene codes for an efflux mechanisms which results in resistance to erythromycin but susceptible to clindamycin.

D-Zone positive Negative

• D zone test for inducible clinda resistance to be performed before reporting clindamycin

• For the D zone test erythromycin and clindamycindisk to be placed 15-26 mm edge to edge on MHA by usual DD test.

• Incubation-16-18hrs at 37c.

• Flatening of the clinda zone between the 2 disk –indicates the isolate has inducible clindamycinresistant because of erm gene

• No flatening the isolate is erythromycin resistant (due to msrA).

• D zone positive- clindamycin resistant

• D zone negative- clindamycin susceptible

• Both erythromycin and clindamycin resistant- clindamycinresistant.

Vancomycin resistant Enterococci:• E. faecium and the E.faecalis are most common resistant to

vancomycin and teicoplanin

• Six different types of vancomycin resistance

• Van A and B are most commonly encountered

• Van A – resistance to both vancomycin and teicoplanin

• Van B - resistance to vancomycin and susceptible to teicoplanin

VRE mechanism

• Alteration to the terminal amino acid residues of the NAM/NAG-peptide subunits

• The D-alanyl-D-lactate variation results in the loss of one hydrogen-bonding interaction

• This loss of just one point of interaction results in a decrease in affinity between vancomycin and peptide

VRE detection

• Disk diffusion

• MIC by broth dilution, agar dilution and E-test

• Vancomycin agar screen plate

• BHIA with 6mg vancomycin can be used for Enterococci and Staphylococci

• Drugs – daptomycin, linezolid, quinipristindalfopristin

High level aminoglycoside resistance

• Enterococci are inherently resistant to the concentration of aminoglycoside producing their use as single agent for treatment of enterococcal infections

• This low level resistance is due to the poor drug uptake by the enterococcal cells.

• However enterococci develop high level aminoglycoside resistance in which the particular aminoglycoside does not demonstrate synergism with the cell wall active agent penicillin or ampicillin.

• High level aminoglycoside resistant in

enterococci is usually the result of enzyamticinactivation of the drugs.

• Detection by

DD – Gentamicin 120µg

MIC - Gentamicin 500µg

ESBL

• The major mechanism of resistant to β-lactam antimicrobial agent in Gram negative bacilli is production of β-lactamaseenzyme because of their increased spectrum activity.

• ESBL are a group of plasmid mediated diverse complex and rapidly evolving enzymes that are posing a major therapeutic challenge in the treatment of hospitalized and community based patients.

• Infections caused by ESBL strains from UTI to life threatening sepsis.

• Β-lactamase these enzymes share the ability to hydrolyse

These cephalosporins include cefotaxime,ceftriaxone, and ceftazidime, as well as monobactam aztreonam.

• ESBL – producing organsims exhibit co-resistance to many other class of antibiotics.

• Because of inoculum effect and substrate specificity –their detection is also major challenge.

• But now CLSI gives the guideline for detection of ESBL in Klebsiella pneumoniae, K.oxytoca, E.coli and Pr.mirabilis.

• ESBL are β-lactamase capable of confering bacterial resistance to the penicillin, I, II and III generation of cephalosporins and aztreonam

β lactamases

Restricted spectrum

β lactamases

ESBL

AmpC β lactamases

CTX-M OXA

Serine MBLClass B

Class A OXAClass D

OthersClassical

TEM-1 & 2, SHV-1

TEM-3, SHV-2

Over 65

types

11, 14, 15, 16,

17

CMY, LAT, FOX

KPC, SME, IMI

23, 24, 40, 51,

58

Carbapenemases

IMP, VIM, NDM

ESBL detection tests - Phenotypic

Screening Confirmatory

1) Disk diffusion method

2) Dilution method – MIC

1) Diffusion methods

- Double disc

- Combination disc

- E-test

2) Dilution method – MIC

Icil

Confirmatory tests

Double disc approximation test

Qualitative only

CLSI combination disc test

Qualitative and quantitative

CTX CTX + CL

CZD CZD + CL

cefotaxime ceftazidime

Amox + clav

CLSI ESBL confirmatory tests interpretation

For E. coli, Klebsiella, and P. mirabilis

MIC test ≥3 two-fold concentration decrease in MIC of cefotaxime/ceftazidime +/- clavulanate 4 μg/ml

Disk test ≥5-mm increase in zone diameter for cefotaxime/ceftazidime +/- clavulanate 10 μg

Icil

AmpC Beta lactamases

• Chromosomal mediated in Enterobacter, Serratia, Citrobacter, Morganella, Providencia

• Plasmid-mediated in E. coli and KlebsiellaEmergence predominantly in community-acquired infections

• Co-resistance to aminoglycosides, SXT, quinolones

TEM, SHV CTX-M OXA AmpC

Cefpodoxime R R R R

Clavulanate S S R R

Cephamycins S S S R

Cefepime R R R S

Drug of Choice

• Carbapenems (Impenem, meropenem)

• Colistin, polymyxin, tigecycline for serious infections

• Co-trimoxazole, nitrofurantoin, fosfomycin, gentamycin, amikacin and inhibitor combinations for uncomplicated infections.

Carbapenemase• Carbapenems – Highest class of beta lactam agent

current available, eg, imipenem, meropenem, ertapenem

• Carbapenem resistance to all beta lactam antibiotics such as penicillin, cephalosporins, monobactams and carbapenems

• Carbapenem resistance due to

Production of carbapenemases

Excess production of ESBL, porin loss, increased efflux pumps

• Carbapenemases are beta lactamase enzyme coded by plasmids

• In GNB most commonly encountered are

• Klebsiella pneumoniae carabapenemase (KPC) -Serine in their active site

• Metallo beta lactamases – Zinc in their active site

• Metallo beta lcatamases – R to all beta lactamsbut S to monobactam

• KPC – R to all beta lactam / beta lactamaseinhibitor

Detection methods

• DD – imipenem, meropenem

• MIC – broth, agar dilutions

• E-test

Modified Hodge test

• Lawn culture of E. coli ATCC 25922 - 1/10 of 0.5 McFarland

• ERT10 μg

• Inoculate cultures as shown in figure

• edge of disk to periphery

CONCLUSION

• AST is very important for the clinician to treat the patient with appropriate antibiotics.

• Formulation of antibiotic policy

• Surveillance of resistance

– In community

– Hospital out breaks

• Lab to upgrade its own good standard

• Ensures accuracy, reliability reproducibility of the test performed.