Antibacterial susceptibility testing
Drug classes
Methods for testing
Laboratory strategies
Basic principles of antimicrobial action
1. Agent is in active form
- pharmacodynamics: structure & route
2. Achieve sufficient levels at site of infection
- pharmacokinetics
Anatomic distribution
Ampicillin
Ceftriaxone
Vancomycin
Ciprofloxacin
Gentamicin
Clindamycin
Norfloxacin
Nitrofurantoin
Serum
CSF
Urine
+
+
+
+
+
+
-
+
+
±
±
-
+
+
+
+
+
+
+
Basic principles of antimicrobial action
3. Adsorption of drug by organism
4. Intracellular uptake
5. Target binding
6. Growth inhibition (bacteriostatic)
or death (bactericidal)
- Resistance can develop at any point
Mechanisms of action
Beta-lactams
Penicillins, cephalosporins, carbapenems
Inhibit cell wall synthesis by binding PBPs
Active against many Gram + and Gram – (varies)
Aminoglycosides
Gentamicin, tobramycin, amikacin, streptomycin
Inhibit protein synthesis (30S ribosomal subunit)
Gram + and Gram – but not anaerobes
Beta-lactams
http://www.life.umd.edu/classroom/bsci424/Definitions.htm
Aminoglycosides
http://gsbs.utmb.edu/microbook/ch011.htm
Mechanisms of action
Fluoroquinolones
Ciprofloxacin, levofloxacin
Inhibit DNA synthesis by binding to gyrases
Active against many Gram + and Gram – (varies)
Glycopeptides
Vancomycin
Inhibit cell wall synthesis by binding precursors
Gram + only
Quinolones
Glycopeptide
http://gsbs.utmb.edu/microbook/ch011.htm
Mechanisms of action
Macrolides-lincosamides
Erythromycin, azithromycin, clindamycin
Inhibit protein synthesis (50S ribosomal subunit)
Most Gram + and some Gram –
Tetracyclines
Tetracycline, doxycycline
Inhibit protein synthesis (30S ribosomal subunit)
Gram + and Gram – and intracellular orgs.
Macrolides
Tetracycline
http://gsbs.utmb.edu/microbook/ch011.htm
Mechanisms of action
Oxazolidinones
Linezolid
Inhibit protein synthesis (50S ribosomal subunit)
Gram + and Gram – including multi-resistant
Streptogramins
Quinupristin/dalfopristin (Synercid)
Inhibit protein sythesis (50S ribosomal subunit)
Primarily Gram + organisms
Linezolid
Streptogramins
http://www.kcom.edu/faculty/chamberlain/Website/Lects/Metabo.htm
Mechanisms of action
Trimethoprim
Sulfonamides
Usually combined (Trimeth/sulfa)
Inhibit different parts of folic acid pathway
affects DNA synthesis
Gram + and many Gram –
http://gsbs.utmb.edu/microbook/ch011.htm
Mechanisms of resistance
Biologic
- physiologic changes resulting in a decrease
in susceptibility
Clinical
- physiologic changes have progressed to a point
where drug is no longer clinically useful
Mechanisms of resistance
Environmentally-mediated
Physical or chemical characteristics that alter the
agent or the organism’s physiologic response to
the drug
pH
anaerobiasis
cations
metabolites
Mechanisms of resistance
Microorganism-mediated
Intrinsic  predictable
Gram neg vs. vancomycin (uptake)
Klebsiella vs. ampicillin (AmpC)
Aerobes vs. metronidazole (anaerobic activation)
Mechanisms of resistance
Microorganism-mediated
Acquired  unpredictable
- this is why we test
- mutations, gene transfer, or combination
Mechanisms of resistance
These factors are taken into account to attempt
to standardize in vitro testing methods.
In vitro methods are not designed to recreate
in vivo physiology.
In vivo physiology affects clinical response such
that in vitro testing cannot be used to predict
clinical outcome.
Mechanisms of resistance
Common pathways
1.
Enzymatic degradation or modification of agent
2.
Decreased uptake or accumulation of agent
3.
Altered target
4.
Circumvention of consequences of agent
5.
Uncoupling of agent-target interactions
6.
Any combination of above
Emergence of resistance
Mixing of bacterial
gene pool
Selective pressure from
excessive antimicrobial
use and abuse
Survival of the fittest
Emergence of resistance
1. Emergence of new genes
- MRSA, VRE, GISA
2. Spread of old genes to new hosts
- Pen resistant GC , GRSA
3. Mutations of old genes resulting in more
potent resistance
- ESBLs
4. Emergence of intrinsically resistant
opportunistic bacteria
- Stenatrophomonas
Methods for detecting resistance
Goal: To determine whether organism
expresses resistances to agents potentially
used for therapy
Designed to determine extent of acquired
resistance
Methods for detecting resistance
Goals of standardization
1.
Optimize growth conditions
2.
Maintain integrity of antimicrobial agent
3.
Maintain reproducibility and consistency
Methods for detecting resistance
National Committee for Clinical Laboratory
Standards (NCCLS)
Name changed to:
Clinical Laboratory Standards Institute
(CLSI)
Methods for detecting resistance
Standardization
Limits:
In no way mimic in vivo environment
Results cannot predict outcome because of:
- diffusion in tissue and host cells
- serum protein binding
- drug interactions
- host immune status and underlying illness
- virulence of organism
- site and severity of infection
Methods for detecting resistance
Standardization
Inoculum size
Growth medium
Incubation atmosphere, temperature, duration
Antimicrobial concentrations used
Methods for detecting resistance
Inoculum preparation
Standardized inoculum size using turbidity
standard
McFarland standard: mixing various volumes of
1% sulfuric acid and 1.175% barium chloride
0.5 McFarland = 1.5 x 108 CFU/mL
Adjust by eye or using instrument
Methods for detecting resistance
Growth media
Mueller-Hinton
pH
Cation conc.
Blood and serum suppl.
Thymidine content
Thickness
Methods for detecting resistance
Incubation conditions
Temperature:
35°C
Atmosphere:
room air (most)
5 – 10% CO2 (fastidious)
Methods for detecting resistance
Incubation time
GNR:
16 – 18 hrs.
GPC:
24 hrs.
Methods for detecting resistance
Selection of antimicrobial agents
Organism identification or group
Acquired resistance patterns of local flora
Testing method used
Site of infection
Formulary
Methods for detecting resistance
Directly measure the activity of one or more
antimicrobial agents against an isolate
Directly measure the presence of a specific
resistance mechanism in an isolate
Measure complex interactions between
agent and organism
Detect specific genes which confer resistance
Methods for detecting resistance
Directly measure antimicrobial activity
Conventional methods
Broth dilution
Agar dilution
Disk diffusion
Commercial systems
Special screens and indicator tests
Conventional methods
Inoculum preparation for manual methods
Pure culture, 4 – 5 isolated colonies,
16 – 24 hrs old
GNR: inoculated into broth and incubated
until reaching log phase
GPC: suspended in broth or saline and
tested directly
Conventional methods
Broth dilution
Various concentrations of agent in broth
Range varies for each drug
Typically tested at doubling dilutions
Minimum inhibitory concentration (MIC):
lowest concentration required to
visibly inhibit growth
Conventional methods
Broth dilution
Microdilution: testing volume 0.05 – 0.1 mL
Macrodilution: testing volume >1.0 mL
Final concentration of organism:
5 x 105 CFU/mL
Conventional methods
Agar dilution
Doubling dilution is incorporated into agar
Multiple isolates tested on each plate
Final amount of organism spotted:
1 x 104 CFU
Visually examine for growth, determine MIC
Conventional methods
Disk diffusion (Kirby-Bauer)
Surface of agar plate seeded with lawn of
test organism
Inoculum: swab from 0.5 McFarland
Disks containing known conc. of agent placed
on surface of plate
Measure diameter of zone of inhibition
Conventional methods
Disk diffusion
Zone sizes have been correlated with MICs
to establish interpretive criteria
Typically, 12 – 13 disks can be placed on
each plate
Conventional methods
Antibiotic gradient diffusion
Agent is applied in gradient to a test strip
Plate is seeded with organism as in KB
Agent diffuses away from strip to inhibit growth
Etest (AB BIODISK, Sweden)
Interpretive categories
Susceptible: agent may be appropriate for
therapy; resistance is absent or clinically
insignificant
Intermediate: agent may be useful if conc.
at site of infection; may not be as useful
as susceptible agent; serves as safety
margin for variability in testing
Resistant: agent may not be appropriate for
therapy; inhibitable dose not acheivable or
organism possesses resistance mechanism
Automated systems
Manual preparation of isolate suspension
Manual – completely automated inoculation
Automated incubation, reading of results
Automated interpretation and data management
MicroScan WalkAway
Dade-Behring
VITEK 2, BioMerieux
Supplemental testing methods
Screening agar
Agar contains known conc. of antibiotic
Growth on agar indicates resistance
Oxacillin screening agar: 6 g/ml oxacillin
Screening of staphylococci
Vancomycin screening agar: 6 g/ml vanco
Screening of enterococci and staphylococci
Supplemental testing methods
Predictor drugs
Staphylococci R to Oxacillin =
R to penicillins, cephalosporins, and imipenem
High level gentimicin R in enterococci =
R to all currently available aminoglycosides
Ampicillin R in enterococci =
R to all penicillin derivatives and imipenem
Direct detection of resistance mechanisms
Beta-lactamase (phenotypic)
Chromogenic substrate incorporated into disk
- color change in presence of enzyme
Usefulness is limited:
Pen R in GC
Amp R in H. flu
Pen R in anaerobes
Direct detection of resistance mechanisms
Extended spectrum beta-lactamase
Mutations in plasmid-encoded beta-lactamases
- hydrolyze extended spectrum cephalosporins
and aztreonam
- more than 100 types have been identified
- isolates are often resistant to other classes
Interpretive criteria available for:
- E. coli, K. pneumoniae, K. oxytoca, P. mirabilis
Direct detection of resistance mechanisms
Extended spectrum beta-lactamase
Screen with aztreonam or cefpodoxime
R = requires confirmatory testing
Confirmatory testing:
Ceftazidime v. ceftaz + clavulanic acid
Cefotaxime v. cefotax + clavulanic acid
KB: >/= 5 mm increase w/ BLI
MIC: >/= 3-fold decr in MIC w/ BLI
Direct detection of resistance mechanisms
Oxacillin R due to PBP2a (phenotypic)
Latex agglutination test to detect altered
PBP in staphylococci
Presence confers resistance to Ox
Depends on expression of protein
Direct detection of resistance mechanisms
Oxacillin R due to PBP2a (genotypic)
PCR to detect mecA gene in staphylococci
Positive not dependent on expression
Direct detection of resistance mechanisms
Inducible clindamycin resistance (D test)
Resistance to macrolides can occur through:
efflux (msrA)
ribosome alteration (erm)
Erythro R
Clinda S
msrA or inducible erm
Erythro R
Clinda R
constitutive erm
L: Erythro, R: Clinda
No resistance
Inducible erm
Efflux
Constitutive erm
Laboratory strategies for testing
Goals of effective strategies include:
Relevance
Accuracy
Communication
Laboratory strategies for testing
Criteria used for assessing relevance:
Clinical significance of isolate
Predictability of susceptibility against drugs
of choice
Availability of reliable standardized methods
Selection of appropriate agents
Laboratory strategies for testing
Clinical significance
Abundance in direct smear
Ability to cause disease in that body site
Colonizer or pathogen?
Body site of isolation
Laboratory strategies for testing
Predictability of susceptibility
Testing not required when susceptibility is
predictable
Pen S in beta-hemolytic streptococci
Ceph S in GC
Clinical requirements can result in exceptions
Laboratory strategies for testing
Availability of standardized methods
Testing cannot be performed if standardized
method does not exist
Method and interpretive guidelines required
Info available for most pathogenic bacteria
Fungi, Nocardia, AFB
Laboratory strategies for testing
Selection of agents
Previously discussed criteria:
Organism ID or group
Acquired resistance patterns
Testing method used
Site of infection
Formulary
Laboratory strategies for testing
Communication
Prompt and thorough review of results
Prompt resolution of unusual results
Augment susceptibility reports with messages
that help clarify and explain potential
therapeutic problems not necessarily
evident by data alone