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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.