Antibiotic
Sensitivity Testing
2020 Update
Margie A. Morgan, PhD,
MT(ASCP), D(ABMM)

Antibiotic Classes
• Beta Lactams –contain a beta lactam
ring in molecular structure of antibiotic
• Penicillins
• Penicillin
• Amoxicillin
• Ampicillin
• Amp/Clavulanate
• Amp/Sulbactam
• Anti-Pseudomonal Penicillins
• Piperacillin/Tazobactam
• Ticarcillin/Clavulanate
• Anti- Staphylococcal Penicillins
• Nafcillin
• Oxacillin
• Cloxacillin
• Dicloxacillin

Antibiotic Classes (2)
• Also beta lactam
antibiotics
Carbapenems
• Imipenem
• Meropenem
• Ertapenem
• Doripenem
• Monobactam
• Aztreonam
• Cephalosporins
• First generation
• Cefazolin
• Second generation
• Cefotetan
• Cefoxitin Cefuroxime
• Third generation
• Ceftriaxone
• Ceftazidime Cefpodoxime
• Fourth generation
• Cefepime Ceftaroline

Antibiotic Classes (3)
• Fluoroquinolones
• Ciprofloxacin
• Levofloxacin
• Moxifloxacin
• Aminoglycosides
• Gentamicin
• Tobramycin
• Amikacin
• Sulfonamide
• Trimethoprim/
sulfamethozaxole
• Macrolides
• Azithromycin
• Clarithromycin
• Erythromycin
• Tetracyclines
• Tetracycline
• Minocycline
• Doxycycline
• Lincosamide
• Chloramphenicol
• Nitrofurans
• Nitrofurantoin

Four Major mechanisms of antibiotic resistance
•Enzymatic cleavage leads to inactivation of an antibiotic
• Active Beta lactamases and amino-glycoside modifying enzymes cleave and destroy
antibiotic
•Altered receptors and binding proteins prevent attachment of
antibiotics to the bacterial surface
• Penicillin binding proteins (PBPs)
• Mechanism for Strep pneumoniae resistance to penicillin and MRSA resistance to
methicillin
•Altered permeability/influx and efflux pumps stop passage of
antibiotic through cell membrane porins – gram negative bacilli
• Pseudomonas aeruginosa resistance to amino-glycosides
•Bypass of a metabolic block/metabolic block imposed by antibiotic
• Enterococcus resistance to TMP/SXT

How are rules established for Susceptibility Testing?
CLSI – Clinical Laboratory Standards Institute publish approved
standards for the testing & reporting of susceptibility results
1. Prepare documents with appropriate antibiotics to test for
each organism
2. Charts outlining interpretation of susceptibility results
3. Quality control standards and proper testing procedures

Methods/Bacteria in Review
Susceptibility testing methods
1. Kirby Bauer disk diffusion (KB)
2. E Test Strip Minimum inhibitory concentration (MIC )
3. Broth dilution Minimum inhibitory concentration (MIC)
Resistant Bacteria of Importance
•MRSA methicillin resistant Staphylococcus aureus
•VRE vancomycin resistant enterococcus
•ESBL Extended Spectrum Beta Lactamase
•CRE Carbapenamase Resistant Enteric Gram negative rods / also
Carbapenamase resistant organisms (CRO)
•Streptococcus pneumoniae
•Neisseria gonorrhoeae

Preparation of Bacteria for all Susceptibility Methods
Pure culture of one organism/ never test a mixed culture
•Log phase growth of bacteria - 16-24 hours old
•Susceptibility methods test for stasis not killing of
bacteria
•Standardized suspension of bacteria needed for testing.
This is accomplished by:
• O.5 McFarland Standard – Barium sulfate solution
prepared in an acid pH that equals the turbidity of 10
8 bacteria/ml
• Alternative method – use spectrophotometer
•Incubation at 35 °C in room air (or CO²) for 18- 24 hours
•If inoculum anount is NOT controlled, false resistance or
susceptible results could occur

Quality Control
Verification: Before testing patient organisms: Must correctly test
multiple QC strains for 20 consecutive days. This is to assure you are
able to perform the tests correctly.
Must use ATCC strains of bacteria (American Type Culture Collection)
with known activity for testing QC. If QC strains are tested and within
appropriate values - you can then do weekly quality control on all lots of
antibiotic cards, disks, plates.
• Data must be recorded and reviewed monthly by supervisor
•If weekly QC results are out of control:
• Immediately repeat/ inform supervisor
• If repeat is OK and you know the source of your error – continue routine testing
• If repeat is NOT OK or if you do not know why you had a failure – must investigate/document/
repeat 5 times to start routine testing. All repeats must be in control
• Must not report results of out of control antibiotic until back in control

Agar Disk Diffusion (Kirby Bauer)
Qualitative Susceptibility Method
•Mueller Hinton agar –with or without blood
• 150 mm plate diameter
• 4mm in depth
• Agar specifically balanced in Ca+ and Mg+,
• if the ions are too high % amino-glycosides test falsely resistant,
• if the ions too low % falsely susceptible amino-glycoside results
•Streak bacteria on plate with cotton tipped swab
•Apply 6mm paper disks that contain a single
antibiotic
•Incubate for 16-24 hrs at 35*C
•Measure zone of diameter of inhibition of growth
(mm) – CLSI charts used to interpret results

Kirby Bauer disk Dispenser
Each cartridge contains a
separate antibiotic
Growth inside a zone is considered
resistance
Measure the diameter
of the zone of inhibition
Watch out for double
Zones /contaminated?
Proteus will swarm
Into a zone

E Test
Quantitative MIC Susceptibility
•Calibrated plastic strips impregnated with
concentration gradient (mcg/ml) of one
antibiotic
•Diffusion gradient created as antibiotic diffuses
into agar in an elliptical shape
•MIC (minimum inhibitory concentration) is
where the ellipse ends on the plastic strip
•Good method for slower growing fastidious
organisms that do not give consistent results on
Kirby Bauer test
.

Broth Dilution minimum inhibitory concentration/MIC
Quantitative Susceptibility Method
•Bacteria inoculum: adjusted to 0.5 McFarland
standard then further diluted to 5x105 organisms /ml in
saline
•Suspension is inoculated into micro titer trays
containing broth growth medium and known 2 fold
dilutions (mcg/ml) of antibiotics
•Each horizontal row is a unique antibiotic
• Growth/resistance leads to turbidity in the broth
•Lowest concentration of antibiotic with No Growth
(clear well)= MIC value.

Broth Dilution Definitions
•MIC = lowest concentration of antibiotic inhibiting growth
•MBC = lowest concentration of antibiotic killing 99.9%
•Antibiotic tolerance – Ratio of MBC/MIC (>=32)
• MBC = 16 MIC= 8 16/8= 2 No Tolerance
• MBC = 128 MIC = 8128/2 = 64 Tolerance*
• *This means bacteria will tolerate the antibiotic and not be killed

Becton-Coulter Microscan walkaway
AST system
BD Phoenix AST systemBiomerieux Vitek2 AST
Automated Identification and
Susceptibility Testing Systems
(AST) – use broth dilution MIC type
testing

Methicillin Resistant Staph aureus (MRSA)
•OLD WAY/ Test for oxacillin (OX) resistance to detect methicillin resistance - it is
more stable for testing in the laboratory
•If OX is resistant - S. aureus is reported as a MRSA
•NEW and better way – If Cefoxitin is resistant report as a MRSA
Cefoxitin is more reliable and a preferred way to confirm MRSA
•All cephalosporin antibiotics are also reported resistant when a MRSA is detected
and should never be used for therapy
•Resistance mechanism for MRSA is penicillin binding proteins (PBPs)
• PBPs bind penicillin and related antibiotics
• The binding prevents disruption of the peptidoglycan synthesis in the Staph aureus cell wall
• PBPs are produced by the mecA gene
• New emerging strain of MRSA produced by mecC gene

Picture: Oxacillin KB disk =
resistant / MRSA
Cefoxitin KB disk Newer
method –more sensitive
screen for MRSA
Picture: Cefoxitin (FOX)
KB = a methicillin sensitive
S. aureus
Methods to Detect MRSA
Oxacillin Testing is no longer
the preferred method for
detection

Clindamycin Induction Test –
The D test
•The D test determines if Staph aureus, including MRSA, is
susceptible to Clindamycin more reliably than testing Clindamycin
resistance by KB or MIC methods
•During antibiotic therapy, Staph aureus isolates resistant to
Erythromycin possess enzymes that can be induced to make the S.
aureus also resistant to Clindamycin
•Clindamycin is often used to treat serious soft tissue infections
with MRSA – so reliable testing necessary

D test Negative-
round Clindamycin zone
Kirby Bauer zone around Clindamycin will be
blunted to form a D if Clindamycin can be
induced by Erythromycin to be resistant – so
called INDUCIBLE RESISTANCE.
Clindamycin should be reported as “resistant
by clindamycin induction” and not used for
therapy
The D Test

Enterococcus
•All Enterococcus are intrinsically resistant to:
• Cephalosporins
• Clindamycin
• Trimethoprim/sulfamethoxazole
•Synergistic antibiotic therapy can be important for the treatment of Enterococcus
• Ampicillin plus Gentamicin is synergistic = increased bacteria killing potential
with combination of two antibiotics that kill by different mechanisms
• Particularly important for endocarditis therapy
•Acquired resistance to vancomycin –
• Plasmid mediated vanA associated with E. faecium
• Plasmid mediated vanB associated with E. faecalis

Extended Spectrum Beta Lactamase [ESBL]
•Enzymes produced by Enteric Gram negative bacilli
• Confer resistance to Cephalosporins, Penicillins and Monobactam
(Aztreonam) antibiotics by opening the beta lactam ring of the antibiotic
and inactivating the antibiotic
• ESBLs do not attack Cephamycin (cefoxitin, cefotetan) or the
Carbapenem antibiotic classes
•Plasmid mediated CTX-M beta lactamases are the most common ESBL
enzymes in the US currently, but many more ESBL types can be found
worldwide
•Therapy for ESBL producing gram negative rods:
• Carbapenems: Imipenem, Meropenem, Doripenem, and
Ertapenem

Detecting ESBL in the laboratory
•Standard of Practice:
• Recently, CLSI established new Cephalosporin MIC values and KB
zone breakpoints to safely detect ESBL activity
• New MIC breakpoints are one to three doubling dilutions lower
than previously used
• New KB zones for susceptible are larger than before
•Molecular testing needed for confirmation of actual enzyme present
(CTX-M)– this is beyond the scope of most clinical laboratories

Why all the fuss about ESBLs?
• GNRs with ESBL phenotypes >=10% in US and the
numbers are increasing
• Limited treatment options
• Carbapenems: meropenem, imipenem, ertapenem
• Risk factors:
• Long hospital stay – particularly in the ICU
• Central lines
• Issues with the intestine
• Long term care facility
• Ventilator assistance

Carbapenemases – CRE and CRO
•CRE = Carbapenamase resistant Enteric / CRO= Carbapenamase resistant organism
•Carbapenem antibiotics currently have the highest spectrum of activity against multi-
drug resistant GNRs
•But the worst scenario has come true – appearance of carbapenem-hydrolyzing-beta-
lactamases which confer resistance to a broad spectrum of beta lactam antibiotics
including carbapenems – making GNRs resistant to most if not all antibiotics
•Two CREs are getting the most attention:
• KPC – “Klebsiella pneumoniae carbapenemase” most common in the US
• NDM-1 – New Delhi metallo-beta-lactamase. Resistance determinants are numerous and great
concern about its spread.
•Infections with CRE/CRO producing GNRs can produce a 50% fatality rate

Most sensitive way to detect a CRE is to perform an Ertapenem
MIC test.
However, all Carbapenem antibiotics (meropenem, imipenem)
usually have MICs in the resistance range for CREs. CROs can
be identified by detecting elevated/resistant MICs to
meropenem or imipenem
Treatment of CRE:
Polymyxins (Colistin and Polymyxin B)
Newer combination antibiotics preferred:
Ceftazidime/avibactam
Ceftolozane/tazobactam
Meropenem/Vabomere
Carbapenemase Resistant CRE
Laboratory Testing

Streptococcus pneumoniae and
resistance to Penicillin (PEN)
• Perform a Penicillin Minimum Inhibitory Concentration (MIC) by E
Test or broth microdilution
•KB method cannot be used to test Penicillin against Strep
pneumoniae because it under predicts resistance to Penicillin
(PEN)
•High level PEN resistance is <=10% in US
• If PEN resistant - antibiotics of choice becomes a 3rd gen
Cephalosporin, vancomycin or quinolone

Neisseria gonorrhoeae (GC)
• Increasing resistance of GC over last two decades
• 1980’s beta lactamase producing strains with Penicillin resistance
• By 2000, the quinolones were resistant due to the acquisition of mutations
by GC that altered the binding sites, preventing Quinolone activity
• Currently Cephalosporins (Ceftriaxone & Cefixime) are the mainstay for
therapy in the US – however resistance to these antibiotics are becoming
common in Asia.
• This resistance due to Penicillin Binding Proteins (PBPs) and over production of efflux
pump mechanism
• Currently susceptibility testing of GC is not performed in most labs
• Detection of resistant strains in the USA could be problematic due to our
reliance on amplification testing for STD diagnosis that only test for the
genetic presence of GC and not for resistance markers