ANTIBIOTIC SUSCEPTABILITY TESTING

(Kirby Bauer Disk Diffusion test)

PURPOSE:

It is used to asses the antibiotic sensitivity of a certain

bacterial isolate.

SPECIMEN :

Pure bacterial isolate from fresh culture plate.

MATERIALS :

1. Nutrient broth for fastidious organisms or sterile saline

for non fastidious organisms.

2. 0.5 Mc Farland standard for adjusting the turbidity of

the inoculums.

3. Vortex mixer for suspension of the inoculum.

4. View box for comparison of broth with standard

5. Mueller-Hinton agar plates unsupplemented for non

fastidious organisms or supplemented with RBCs in a

concentration of 5% for fastidious organisms (90-mm

diameter for seven disks; 150-mm diameter for a

maximum of 12 disks) from a lot that gives a

satisfactory quality control results. The PH must be 7.2

to 7.4, and the depth must be approximately 4 mm.

1

6. Non-CO2 35- 37˚C incubator for non fastidious

organisms or 5% CO2 incubation for H.inf -

S.pneumoniae- N.meningititis

QUALITY CONTROL

Antibiotic discs for susceptibility testing are checked

weekly utilizing appropriate ATCC reference strains. In

addition, QC testing will be performed anytime when

antibiotic with a new lot number is used repeat the testing.

Document any corrective action in the QC log book. The

discs tested for QC must be the same discs used with the

patient specimens. Tolerance limits for antimicrobial potency

are based on CLSI guidelines. If the zone range limits are

exceed, the Lab Director must be immediately notified and

no sensitivity results will be reported.

E coli ATCC 25922Pseudomonas aeroginosae ATCC 27853S. aureus ATCC 29213

ALSO QC STRAINS FOR ESBL & FASTIDIOUS ORGANISMS MUST BE INCLUDED

QC ORGANISMS MAINTENANCE :

Avoid repeated subculture

Store stock isolates at -60C or below

Prepare working culture weekly & stored at -20C

2

PROCEDURES

1. Preparation of inoculum :

a. With a sterile wire loop, touch the top of two to

five similar- appearing, well-isolated colonies on

an agar plate culture according to the size of

colonies as follows: large colonies as

citrobacter touch only the quarter of its size,

small colonies as strept touch five colonies,

while moderate sized colonies touch only

two colonies.

b. Emulsify them in 5mL of sterile physiological saline

or nutrient broth with the help of vortex.

c. The turbidity of the emulsification is adjusted to

0.5Mc Farland standard. Turbidity is matched

against a printed card or sheet of paper in a good

light.

d. Within 15 minutes of adjusting the turbidity of the

inoculums suspension, add the suspension to the

plate by pouring the suspension on the surface of

the agar plate, and then discard the excess in

waste container which contain a disinfectant

Replace the lid of the dish .Allow at least 5

3

minutes but no longer than 15 minutes for the

surface to dry before adding the antibiotic disks

2. Testing of antibiotics:

a. Place the appropriate antimicrobial-impregnated

disks with specific concentration according to

(CLSI recommendation, age, pregnancy, inpatient

vs outpatient, type of specimens ) on the surface

of the agar, using forceps. Disks must be evenly

distributed on the agar so that they are no closer

than 25 mm from center to center and about 15

mm from the edge of the agar plate.

b. Gently tamp each disk down onto the agar to

provide uniform contact.

c. Within 15 minutes of applying the disks, invert the

plate and incubate it aerobically (ambient air) at

37˚C for 16-18 hours. Examine the plates after the

overnight incubation except for staph & strept up

to 24 hours

INTERPRETATION

With the use of a ruler or a template, the zones of

complete growth inhibition around each of the disks are

carefully measured to within the nearest millimeter; All 4

measurement are made by the unaided eye while viewing

the back of the petri dish with reflected light against a black,

non reflecting background. The plates should be viewed from

a directly vertical line of sight to avoid any parallax that may

result in misreading.

An interpretive correlate (susceptible, moderately

susceptible, intermediate or resistant) is provided by

reference to published CLSI guidelines.

LIMITATIONS

1- Do not move a disk once it has contacted the agar,

because some of the drug diffuses almost immediately.

2- Susceptibility plates prepared with blood must be

viewed from the agar surface and measurements made

with the cover of the Petri dish removed.

3- Zones that fall into the intermediate range should be

considered equivocal; if therapy with the drug is desired, a

dilution susceptibility test should be performed to clarify

the issue.

4- When testing staphylococci against methicillin or

oxacillin or enterococci against vancomycin, incubation

should be for 24 hours.

5

5- Motile organisms such as Proteus mirabilis or P.vulgaris

may swarm when growing on agar surfaces, resulting in a

thin veil that may penetrate into the zones of inhibition

around antimicrobial agent susceptibility disks. This zone of

swarming should be ignored; the outer margin, which is

usually clearly outlined, should be measured. Similarly, with

sulfonamide disks, growth may not be completely inhibited

at the outer margin, resulting in a faint veil, where 80% or

more of the organisms are inhibited. The clear zone of ~

80% inhibition should be read as the zone diameter.

6- Presence of distinct colonies within the zone of inhibition

(2ry colonies) represent either mutant of the same species

that are more resistant to the antimicrobial agent than the

major portion of the bacterial strain being tested or the

culture is not pure and the separate colonies are of a

different species. If it is determined that the separate

colonies represent a variant of a mutant strain, the bacterial

species being tested must be considered resistant. If it is

determined to be a different species, return to the culture

Petri dish and realize whether it is a missed colony or a

contamination. If missed, do a separate antibiogram for the

isolate.

7- When there is overlapping between adjacent agent

zones, zones extend beyond the margin of the Petri dish

or oval(elliptical) zones;, the test must be repeated with

more careful placement of the antimicrobial agent disks

so that overlapping will not occur .When the plate is

6

streaked poorly , this will lead to indistinct zones and the

test must be repeated

CHOICE OF ANTIBIOTICS IN ANTIBIOGRAM:

Drugs are listed by CLSI in 4 groups:

1- GROUP A: Testing & reporting against all isolate.

2- GROUP B: Testing when isolate is resistant to groupA.

3- GROUP C: Supplemental or alternative agent that can

be tested &reported in institutions that harbor resistant

strains.

4- GROUP U: Agents that should be tested & reported only

on isolates from urine.

5- Group O :

6- Group I :

Protocol of antibiotics choice in mic. lab

IN THE FIRST DAY GROUP A & B (GROUB 1) are tested in non urine isolates & (GROUP U) in urine isolates.

IN THE SECOND DAY

7

GROUP C ( GROUP 2) for H.Infleuanza & Enterobacteriacae & antimicrobial combinations will be done .

IN THE THRID DAY Another combination will be tested in multi resistant strains

Antimicrobial agents with FDA clinical indication that should be considered for routine testing

Acinetobacter Fortum (CAZ) Tienam (IPM) or Meronam(MEM) Unasyn (SAM) Ciprofloxacin(CIP) or Levofloxacin(LEV)

Or ofloxacin ( OFX) Gentamycin (CN) or tobramycin (TOB) or

amikin (AK) SUTRIM ( SXT) Sulperazone (SCF) Cefotaxime (CTX) or Rocephine (CRO) Doxycycline (Do) or Tetracycline (TE) Tazocin (TZP)

Polymyxin local only in eye, ears

8

H. Influenza Nisseria Gonorrhea Ampicillin (AMP) Sutrim (SXT) Unasyn (SAM) Cefuroxime (CXM) Cefotaxime (CTX) Rocephine (CRO) Fortum (CAZ) Azithromycin ( AZM) Augmentin ( AMC ) Cefopodoxime

( CPD) Ciprofloxacin ( CIP )

or Levofloxacin LEV Tienam or meronam

Cefopodixime( CPD) Cefotaxime( CTX) Rocephine (CRO) Fortum (CAZ) Ciprofloxacin( CIP) Ofloxacin (OFX)

Penicilin ( P) Tetracyclin (TE)

NB : Testing of B lactamase is mandatory for both isolates using either penicillin disc or nitrocefin sticks .

Burkholderia Stenotrophomonas Sutrim (SXT) Sutrim (SXT)

Fortum (CAZ ) levofloxacin

Meronam ( MEM) minocycline

minocycline

9

. Haemolytic strept (pyogens) Strep. Viridance S. pneumoniae Ampicillin (AMP) Pencillin (P) Erythromycin(E)/ AZM Clindamycin(Cd) Maxipime (FEP) Cefotaxime(CTX) or

Rocephine (CRO) Vancomycin (VA) Levofloxacin(LEV) or

Ofloxacin (OFX) Bacitracin BC

Ampicillin(AMP) Pencillin P(MIC) only Maxipime (FEP) Cefotaxime(CTX) Rocephine(CRO) Vancomycin(VA) Erythromycin /AZM Clindamycin (Cd)

Erythromycin(E)/AZM Oxacillin (ox) testing

penicillin Sutrim (SXT) Clindamycin(Cd) Levofloxacin(LEV) Ofloxacian (OFX) Tetracycline (TE) Vancomycin (VA) Optochine OP

N.B If oxacillin sensitive S.pneumoniae report blindly all penicillins & cephalosporins sensitive but if resistant MIC for 3rd generation cephalosoprins is mandatory

10

Enterococci ( non urine) Staph ( non urine ) Penicillin(P) Ampicillin (AMP) Vancomycin (VA) CN 120µg (high level

screen) or Streptomycin Erythromycin or

Azithromycin Tetracycline ( TE)

Cefoxitin (Fox 30g) Penicillin (P) if sensitive

report all penicillins cephalospoines & carbapenems are sensitive approved by FDA

Sutrim (SXT) Clindamycin(Cd) test of MLS

resistance is recommended Azithromycin(AZM)or

Erythromycin (E) Vancomycin(VA) CIP or OFX or LEV in MSSA

only DO or TE Gentamycin ( CN) Caphalothin ( CF )

Enterococci( urine )

Ciprofloxacin(CIP) Levofloxacin Norfloxacin

Furadantin (F) Tetracycline(TE) P AMP Vancomycin

Staph (urine )

Norfloxacin(NOR) Ofloxacin(OFX) Levofloxacin FOX P VA Furadantin (F) Sutrim (SXT)

Quinolones are not recommended in ttt of MRSA

11

ESBL confirmation in Klebsiella , Ecoli & Proteus (blood isolates only) :

DISC APPROXIMATION TEST

AMC better or SAM ( at the center of the plate)

CPD alone or CAZ& CTX together ( 2.5cm around AMC from center to center)

FOX 30µg ( beside cephalosporin)

TZP OR SCF

FEP ( mandatory)

IPM OR MEM ( beside cephalosporin )

OFX OR LEV / NOR in urine only

CN OR AK

In urine add furadantin

12

CHART FOR ESBL SCREENING & CONFIRMATION Furadantin in urine only

FOX30µg Or

FEP

OFX OR LEV

/NOR in urine only

CN or AK/Furadantin

in urine

TZP or SCF

IPM OR MEM

CPD

CAZ CTX, CFP

AMC MAINLY OR SAM OR

13

Proteus & Enterobacter , Citrobacter

Pseudomonas

Gentamycin (CN) or Tobramycin (TOB) or Amikin (AK)

Unasyn (SAM) or Augmentin (AMC) Tazocin(TZP) Cefepime ( FEP ) cefotrioxne (CRO)or

Cefotaxime (CTX) Cefobid (CFP) Sulperazone (SCF) Ciprofloxacin ofloxacin or Levofloxacin (LEV) Tienam (IPM)or

Meronam (MEM) Sutrim (SXT) Tetracycline (TE)

Fortum (CAZ) Gentamycin (CN) orAmikin (AK) or Tobramycin Tazacin (TZP) Cefobid (CFP) CIP or LEV IPM or MEM SCF FEP

NB: fortum is 3rd generation cephalosporin with strong antipseudomonal activity while rocephine / claforan have weak antipseudomonal activity .

Coryneform bacteria (Diphtheroid)o Sutrimo Penicillino Levo/ofloxo Erythromycin/azythromycino Vancomycino Tetracycline/doxyyclineo Augmentin/ unasyn

14

Gram negative bacilli other than Ecoli & Klebsiella in urine :

Norfloxacin (NOR) OFX/LEV/CIP Furadantin (F) Lomefloxacin Sutrim (SXT) Cefobid ( CFP) Augmentin (AMC ) Gentamycin ( CN ) Carbencillin Tazocin FEP SCF

15

CLSI recommendations:

1. The following antimicrobial agent may be appear active in vitro but are not

effective clinically and should not reported as susceptible:

Salmonella and Shigella

1st and 2nd generation cephalosporins and aminoglycoside

MRSA

Penicillin resistant oxacillin sensitive staph

All . Lactam, carpenams,

All penicillins except B lactamase inhibitors, cephms, & carbapenems

Enterococcus Aminoglycoside (except high conc.) cephalosporins, clindamycin, SXT

Yersinia . Lactam

Listeria

ESBL

Ampicillin resistant enteroccoci

Penicillin (Oxacillin) resistant pneumoniae

Cephalospornis

Penicillin ,cephalosporins & azactam

Penicillin ,B lactamase inhibitor ( AMC, SAM) & carbapenems the mechanism is altered PBPs

Penicillins ,cephalosporins ,carpenams except 3rd generation cephalosporins must do MIC

2. Warning (CSF): The following antimicrobial agent

should not be routinely reported:

a. Agents ad. By oral routes.b. 1st and 2nd gener ceph. except (CXM).c. Clindamycin.

16

d. MacroLides.e. Tetracyclines.f. Fluroquinolones.

3. Susceptibility testing of penicillins and other lactams

approved by FDA for treatment of strep. Pyogenes and

agalactiae is not necessary for clinical purposes.

Recommendation P/AMP/cefazolin/clindamycin/

erythromycin.

4. P.mirabilis should be added to E.coli and

K.pneumoniae in screening for ESBL in bacterimic

isolates only (blood) because reports of ESBL in non

bacterimic isolates have been relatively rare due to low

frequency of plasmid conjugate.

5. Levofloxacin should be used for stenotrophomonas

with SXT.

6. Susceptibility testing is not recommended for

S.saprophyticus in urinary isolates NOV (R) <16mm.

7. Lab should identify S.lugdunensis (an uncommon) but

one cause of endocarditis: (PYR test +ve and ornithine

de carboxylase +ve).

8. Screening of MRSA by cefoxitin 30 or 10g by disc

diffusion while by oxacillin MIC by E test or broth dilution.

For CONS, the cefoxitin disk test has greater specificity

than oxacillin and equal sensitivity, although it may miss

some strains of mecA-detection and the latex test for

17

PBP2a are the most accurate predictors of mecA-

mediated resistance.

9. For organisms (campy, corynebacterium, bacillus

spp.) consultation with an infectious disease specialist is

recommended for guidance in determining the need for

susceptibility testing and in the interpretation of results,

published reports in medical literature and current

consensus recommendations for therapy of uncommon

isolates, may obviate the need for testing. If necessary a

dilution method usually will be the most appropriate

testing method and this may require submitting the

organism to a reference lab.

N B :

Cephalothin CF/CF/CL is the disc

representative for the 1st, 2nd gener cephal

and cefopodoxime

Tetracycline: is representative for Do,

minocyclin.

Erythromycine: is representative for

macrohide.

10. Oxacillin screening disk diffusion used to detect high

rate of penicillin resistance in S.pneumoniae (>20mm

susceptible, <19mm (do MIC testing) and correlate

with the body site it is collected from.

18

11. S.pneumoniae isolates from CSF, it is recommended

testing penicillin, cefotaxime ceftrioxone, meronam and

vancomycin by broth dilution method as disc diffusion

with carbapenems or cephalosporines for S.pneumoniae

do not exist. But for non life threatening infections,

agents to be consider are penicillin, erythromycin sutrim,

by broth and disc diffusion method.

12. Staph isolates that are resistant to erythromycin but

susceptible to clindamycin should be tested for inducible

resistnace to clindamycin (MLS) resistance mediated by

"erm" gene using the D-zone approximation test with

closely approximated erythromycin and clindamycin test.

13. Strept viridans any isolate from a sterile body site or

implicated in a serious infection as endocarditis should

be tested for penicillin susceptibility and cephalosporins

especi especially 3rd generation as some viridans may

exhibit relative resistance. Vancomycin is the

recommended alternative to lactam Abs.

14. For fecal isolates of salmonella and shigella: (ampicillin

, fluroquinolones , SXT (only).while in Extra intestinal

isolates : Chloramphenicol , 3rd generation cephalosporins.

15. As regards ESBL detection:

Due to variable affinity of these enzymes for

diffusion subs and inoculum effect, some ESBL 19

producing organism with 3rd generation cephalosporins

may result in clinical failure if infection is (outside the

urinary tract).

Testing of cephamycins is recommended in ESBL

producing isolates.

Cefpodoxime and ceftazidime have been proposed

as indictors of ESBL production as compared to

cefotaxime and ceftrioxone.

These enzymes can be induced by certain Abs,

AAs, or body fluids.

It is possible for one specimen to contain both

ESBL producing and non ESBL producing cells of the

same species. So, it must test several colonies for a

primary culture plate.

Latest guidelines recommended screening of ESBL

with a MIC 2mg/dL against cefpodoxime ceftazidime,

aztreonam, ceftaxime or ceftrixone.

Three indicators of ESBL:

An 8 fold reduction in MIC in the presence of

clavulonic acid by broth dilution method.

Potentiation of the inhibitor zone by clavulonic

acid >5mm in diameter of inhibition by disc

diffusion.

Disc approximation test by using of cefoxitin

(inducer) placed at a distance of 2.5cm from

20

cephalosporin disc flattening of the zone of

inhibition of cephalosporin disc towards inducer

disc >1mm.

As regards treatment of ESBL carbapenens are the

most effective and reliable as they are highly

resistant to hydrolytic activity of all ESBL enzymes

due to trans-6-hydroxy ethyl group.

Meronam is the most active with MICs generally

lower than those of IPM (0.03-0.12mg/ml vs 0.06-

0.5mg/ml).

Also ESBL activity is inhibited by clavulonic acid,

the only infections that can be treated safely with

lactamase inhibitor are those involving the urinary

tract in which the concentration high enough to

counteract the hydrolytic activity of ESBL.

Clavulonic acid appears more efficient than

sulbactam it takes about eight times more to obtain

a protective similar to that by C.acid.

Plasmids responsible for ESBL production tend to be large and carry resistance to several agents an important limitation in the design of treatment. The most frequent co-resistance are aminoglycosides, flouroquinolones , TE, chloramphenicol and sutrim

21

Issue No/Revision No:Ain Shams University

HospitalsIssue date:Revision date:Copy number:

Code No:Main LaboratoriesPage of

ANTIMICROBIAL COMBINATION BY DIFFUSION METHODS:

Disk approximation test :

Principle:

This method has been explored to assess primarily in a

qualitative fashion the interaction of antimicrobials as they

diffuse through agar plates seeded with a test organism.

Advantages:

Simple.

The use of readily available materials (discs and

Muller Hinton agar).

Disadvantages:

Qualitative method only.

Low sensitivity and specificity compared to dilution

methods.

i.e.: The results of this test may differ from results obtained when the same agents and organism are tested in liquid media.

22

Procedure: This technique uses the same standard inoculums

and Muller-Hinton agar as a routine Bauer-Kirby

susceptibility test.

To assess possible interactions between two drugs

(A and B) disks containing these drugs are placed on a

plate that has been inoculated with a tested organism.

The distance by which the disks are separated

may be varied, but it should generally be equal to or

slightly greater than the sum of the radii of the zones of

inhibition of the drugs when examined alone (mostly

15mm from centre to centre).ONLY FIVE COMBINATION

ARE TESTED IN THE 100 mm PLATE

After overnight incubation (16-18hrs) at 37C the

plate are ready for examination.

23

Issue No/Revision No:Urine Microbiological

ExaminationAin Shams University

Hospitals Issue date:Revision date:Copy number:

Code No:Main LaboratoriesPage of

Example of antibiotic combinations used for multi resistant organisms "by Dilution methods":1. Pseudomonas:

Bactericidal:

Ciprofloxacin and tienam (CIP and IPM).

Ciprofloxacin and Amikin (CIP and AK).

Ciprofloxacin and Azactam (CIP and ATM).

Ciprofloxacin and Fortum (CIP and CAZ).

Levofloxacin and Maxipime (LEV and FEP).

Ciprofloxacin and Maxipime (CIP and FEP).

Levofloxacin and Meronam (LEV and MEM).

24

Ciprofloxacin and Tazocin (CIP and TZP).

Levofloxacin and Tazocin (LEV and TZP).

Levofloxacin and Gentamycin (LEV and CN).

Tazocin and Gentamycin (TZP and CN).important

Tazocin and Tienam (TZP and IPM).

Bacteriostatic:

Augmentine and Ampicillin (AMC and AMP).not used

Vanocomycin and Carbencillin (VA and Pip).not used

Azactam and Maxipime (ATM and FEP).

2. Acinetobacter:

Bactericidal :

Doxycyclin and Amikin (Do and AK).

Ciprofloxacin and Fortum (CIP and CAZ).

Ciprofloxacin and Meranam (CIP and MEM).

Ciprofloxacin and Azactam (CIP and ATM).

Tazocin and Gentamycin (TZP and CN).

Ciprofloxacin and Tazocin (CIP and TZP).

Bacteriostatic :

Tienam and Amikin (IPM and AK).

Unasyn and Amikin (SAM and AK).

3. Enterobacteriaceae:

o Tazocin and Amikin or Gentamycin (TZP and AK or

CN).

25

o Cefotaxim and Amikin or Gentamycin (CTX and AK

or CN).

o Azactam and Tienam (ATM and APM).

o Azactam and Maxipime (ATM and FEP).

o Ceftazidime and Oflaxocin (CTZ and OFO).

o Cefoxitin and Amikin (FOX and AK).

o Ciprofloxacin and Fortum (CIP and CAZ).

o Ciprofloxacin and Tazocin (CIP and TZP).

4. Proteus:

o Tazocin and Amikin (TZP and AK).

o Tienam and Amikin (IPM and AK).

5. Enteroccoci:

.lactam (penicillin) and amino glycoside.

(gentamycin )

Glycopeptide (VA or TEC) and aminoglycoside.

Teinam and Teicoplanin (IPM and TEC).

Tazocin and Gentamycin (TZP and CN).

Tazocin and Ciprofloxacin (TZP and CIP).

Glycopeptide and .lactam (TEC and P).

Ciproflxocin and Vancomycin or Penicillin (CIP and

VA).

26

Ciproflxocin and Ampicllin (CIP and AMP).

27

28