Antibacterial susceptibility testing

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


Fluoroquinolones

Ciprofloxacin, levofloxacinInhibit DNA synthesis by binding to gyrasesActive against many Gram + and Gram – (varies)

Glycopeptides

VancomycinInhibit cell wall synthesis by binding precursorsGram + only


Quinolones

Glycopeptide


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.


Macrolides

Tetracycline


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


Trimethoprim

Sulfonamides

Usually combined (Trimeth/sulfa)Inhibit different parts of folic acid pathway

affects DNA synthesisGram + and many Gram –


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


Environmentally-mediated

Physical or chemical characteristics that alter theagent or the organism’s physiologic response tothe drug

pHanaerobiasiscationsmetabolites


Microorganism-mediated

Intrinsic predictable

Gram neg vs. vancomycin (uptake)

Klebsiella vs. ampicillin (AmpC)

Aerobes vs. metronidazole (anaerobic activation)


Microorganism-mediated

Acquired unpredictable- this is why we test- mutations, gene transfer, or combination


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.


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


Goals of standardization

1. Optimize growth conditions

2. Maintain integrity of antimicrobial agent

3. Maintain reproducibility and consistency


National Committee for Clinical LaboratoryStandards (NCCLS)

Name changed to:

Clinical Laboratory Standards Institute(CLSI)


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


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


Growth media

Mueller-HintonpHCation conc.Blood and serum suppl.Thymidine contentThickness


Incubation conditions

Temperature: 35°C

Atmosphere: room air (most)5 – 10% CO2 (fastidious)


Incubation time

GNR: 16 – 18 hrs.

GPC: 24 hrs.


Selection of antimicrobial agents

Organism identification or group

Acquired resistance patterns of local flora

Testing method used

Site of infection

Formulary



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


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


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


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


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


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


Disk diffusion

Zone sizes have been correlated with MICsto establish interpretive criteria

Typically, 12 – 13 disks can be placed oneach plate


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


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


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


Oxacillin R due to PBP2a (phenotypic)

Latex agglutination test to detect alteredPBP in staphylococci

Presence confers resistance to Ox

Depends on expression of protein


Oxacillin R due to PBP2a (genotypic)

PCR to detect mecA gene in staphylococci

Positive not dependent on expression


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


Criteria used for assessing relevance:

Clinical significance of isolate

Predictability of susceptibility against drugsof choice

Availability of reliable standardized methods

Selection of appropriate agents


Clinical significance

Abundance in direct smear

Ability to cause disease in that body site

Colonizer or pathogen?

Body site of isolation


Predictability of susceptibility

Testing not required when susceptibility ispredictable

Pen S in beta-hemolytic streptococciCeph S in GC

Clinical requirements can result in exceptions


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


Selection of agents

Previously discussed criteria:

Organism ID or groupAcquired resistance patternsTesting method usedSite of infectionFormulary


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

Documents

Antibacterial susceptibility testing