antibacterial susceptibility testing
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
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
Serum CSF Urine
Ampicillin + + +Ceftriaxone + + +Vancomycin + ± +Ciprofloxacin + ± +Gentamicin + - +Clindamycin + - -Norfloxacin - - +Nitrofurantoin - - +
Anatomic distribution
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, carbapenemsInhibit cell wall synthesis by binding PBPsActive against many Gram + and Gram – (varies)
Aminoglycosides
Gentamicin, tobramycin, amikacin, streptomycinInhibit protein synthesis (30S ribosomal subunit)Gram + and Gram – but not anaerobes
http://www.life.umd.edu/classroom/bsci424/Definitions.htm
Beta-lactams
http://gsbs.utmb.edu/microbook/ch011.htm
Aminoglycosides
Mechanisms of action
Fluoroquinolones
Ciprofloxacin, levofloxacinInhibit DNA synthesis by binding to gyrasesActive against many Gram + and Gram – (varies)
Glycopeptides
VancomycinInhibit cell wall synthesis by binding precursorsGram + only
http://gsbs.utmb.edu/microbook/ch011.htm
Quinolones
Glycopeptide
Mechanisms of action
Macrolides-lincosamides
Erythromycin, azithromycin, clindamycinInhibit protein synthesis (50S ribosomal subunit)Most Gram + and some Gram –
Tetracyclines
Tetracycline, doxycyclineInhibit protein synthesis (30S ribosomal subunit)Gram + and Gram – and intracellular orgs.
http://gsbs.utmb.edu/microbook/ch011.htm
Macrolides
Tetracycline
Mechanisms of action
Oxazolidinones
LinezolidInhibit 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
http://www.kcom.edu/faculty/chamberlain/Website/Lects/Metabo.htm
Streptogramins
Mechanisms of action
Trimethoprim
Sulfonamides
Usually combined (Trimeth/sulfa)Inhibit different parts of folic acid pathway
affects DNA synthesisGram + and many Gram –
http://gsbs.utmb.edu/microbook/ch011.htm
Mechanisms of resistance
Biologic
- physiologic changes resulting in a decreasein susceptibility
Clinical
- physiologic changes have progressed to a pointwhere drug is no longer clinically useful
Mechanisms of resistance
Environmentally-mediated
Physical or chemical characteristics that alter theagent or the organism’s physiologic response tothe drug
pHanaerobiasiscationsmetabolites
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 attemptto standardize in vitro testing methods.
In vitro methods are not designed to recreatein vivo physiology.
In vivo physiology affects clinical response suchthat in vitro testing cannot be used to predictclinical 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 bacterialgene pool
Selective pressure fromexcessive 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 organismexpresses resistances to agents potentiallyused for therapy
Designed to determine extent of acquiredresistance
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 LaboratoryStandards (NCCLS)
Name changed to:
Clinical Laboratory Standards Institute(CLSI)
Methods for detecting resistance
Standardization
Limits:In no way mimic in vivo environmentResults 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
Inoculum preparation
Standardized inoculum size using turbiditystandard
McFarland standard: mixing various volumes of1% 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-HintonpHCation conc.Blood and serum suppl.Thymidine contentThickness
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
Methods for detecting resistance
Directly measure the activity of one or moreantimicrobial agents against an isolate
Directly measure the presence of a specificresistance mechanism in an isolate
Measure complex interactions betweenagent and organism
Detect specific genes which confer resistance
Methods for detecting resistance
Directly measure antimicrobial activity
Conventional methodsBroth dilutionAgar dilutionDisk 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 incubateduntil reaching log phase
GPC: suspended in broth or saline andtested 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 tovisibly 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 oftest organismInoculum: swab from 0.5 McFarland
Disks containing known conc. of agent placedon surface of plate
Measure diameter of zone of inhibition
Conventional methods
Disk diffusion
Zone sizes have been correlated with MICsto establish interpretive criteria
Typically, 12 – 13 disks can be placed oneach 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 fortherapy; resistance is absent or clinicallyinsignificant
Intermediate: agent may be useful if conc.at site of infection; may not be as usefulas susceptible agent; serves as safetymargin for variability in testing
Resistant: agent may not be appropriate fortherapy; inhibitable dose not acheivable ororganism 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 WalkAwayDade-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 oxacillinScreening of staphylococci
Vancomycin screening agar: 6 g/ml vancoScreening 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 GCAmp R in H. fluPen 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 cefpodoximeR = requires confirmatory testing
Confirmatory testing:Ceftazidime v. ceftaz + clavulanic acidCefotaxime v. cefotax + clavulanic acid
KB: >/= 5 mm increase w/ BLIMIC: >/= 3-fold decr in MIC w/ BLI
Direct detection of resistance mechanisms
Oxacillin R due to PBP2a (phenotypic)
Latex agglutination test to detect alteredPBP 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 msrA or inducible ermClinda S
Erythro R constitutive ermClinda R
L: Erythro, R: Clinda
No resistance Efflux
Inducible erm 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 drugsof 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 ispredictable
Pen S in beta-hemolytic streptococciCeph S in GC
Clinical requirements can result in exceptions
Laboratory strategies for testing
Availability of standardized methods
Testing cannot be performed if standardizedmethod does not exist
Method and interpretive guidelines required
Info available for most pathogenic bacteriaFungi, Nocardia, AFB
Laboratory strategies for testing
Selection of agents
Previously discussed criteria:
Organism ID or groupAcquired resistance patternsTesting method usedSite of infectionFormulary
Laboratory strategies for testing
Communication
Prompt and thorough review of results
Prompt resolution of unusual results
Augment susceptibility reports with messagesthat help clarify and explain potentialtherapeutic problems not necessarilyevident by data alone