Transcript Resistant - Pathology
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 in vivo methods are not designed to recreate physiology.
In vivo that physiology affects clinical response such in vitro testing cannot be used to predict clinical outcome.
Mechanisms of resistance
Common pathways
1.
2.
3.
4.
5.
6.
Enzymatic degradation or modification of agent Decreased uptake or accumulation of agent Altered target Circumvention of consequences of agent Uncoupling of agent-target interactions 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 Results cannot predict outcome because of: - diffusion in tissue and host cells - serum protein binding - drug interactions environment - 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 10
8
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% CO
2
(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 10
5
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 10
4
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 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 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