Antimicrobial Susceptibility Testing Flashcards
What three classes of antimicrobials fall under the category of beta-lactams?
- Pencillins
- Cephalosporins
- Carbapenems
What antimicrobial class is penicillin in?
Narrow spectrum penicillins
What antimicrobial class is oxacillin in?
Penicillins (beta-lactamase resistant)
What antimicrobial class is piperacillin in?
Broad-spectrum penicillin (ureidopenicillins)
What antimicrobial class is cefazolin in?
Cephalosporins (1st generation)
What antimicrobial class is cefuroxime in?
Cephalosporins (2nd generation)
What antimicrobial class is ceftazidime in?
Cephalosporins (3rd generation)
What antimicrobial class is cefepime in?
Cephalosporins (4th generation)
What antimicrobial class is imipenem in?
Carbapenems (beta-lactams)
What antimicrobial class is meropenem in?
Carbapenems (beta-lactams)
What antimicrobial class is doripenem in?
Carbapenems (beta-lactams)
What antimicrobial class is vancomycin in?
Glycopeptides
What antimicrobial class is ciprofloxacin in?
Floroquinolones
What antimicrobial class is levofloxacin in?
Fluoroquinolones
What antimicrobial class is gentamicin in?
Aminoglycosides
What antimicrobial class is tobramycin in?
Aminoglycosides
What antimicrobial class is is amikacin in?
Aminoglycosides
What antimicrobial class is tetracycline in?
Tetracyclines
What antimicrobial class is doxycycline in?
Tetracyclines
What antimicrobial class is clindamycin in?
Under its own category
What antimicrobial class is is erythromycin in?
Macrolides
What antimicrobial class is trimethoprim-sulfamethoxazole (Bacitrim) in?
Antimetabolites
Penicillin
- Spectrum of activity
- Mechanism of action
- Against GP and GN (changes depending upon class)
- Inhibits cell wall synthesis
Cephalosporins (4th generation)
- Spectrum of activity
- Mechanism of action
- GNRs
- Inhibits peptidoglycan
Tetracyclines
- Spectrum of activity
- Mechanism of action
- Broad spectrum: GP, GN, mycoplasma, chlamydiae, rickettsiae
- Inhibits protein synthesis
Clindamycin
- Spectrum of activity
- Mechanism of action
- Broad spectrum (aerobic GP + anaerobes)
- Inhibits protein synthesis
Fluoroquinolones
- Spectrum of activity
- Mechanism of action
- Broad spectrum: GP, GN
- Inhibits DNA synthesis
Cephalosporins (2nd generation)
- Spectrum of activity
- Mechanism of action
- GPC, some GPR
- Inhibits peptidoglycan synthesis
Antimetabolites
- Spectrum of activity
- Mechanism of action
- Specific clinical uses…UTI, S. maltophilia
- Inhibits folic acid synthesis
Macrolides
- Spectrum of activity
- Mechanism of action
- Broad spectrum
- Inhibits protein synthesis
Cephalosporins (3rd generation)
- Spectrum of activity
- Mechanism of action
- GNR, some GPC
- Inhibits peptidoglycan synthesis
Aminoglycosides
- Spectrum of activity
- Mechanism of action
- Active against GNRs and S. aureus
- Inhibits protein synthesis
Cephalosporins (1st generation)
- Spectrum of activity
- Mechanism of action
- GPC
- Inhibits peptidoglycan synthesis
Glycopeptides
- Spectrum of activity
- Mechanism of action
- GP only
- Inhibits peptidoglycan synthesis
How many interactions occur b/w a patient, an organism, and an antimicrobial?
Six
Substance naturally produced by living organisms such as bacteria and fungi and able, in a dilute solution, to inhibit or kill another microorganism
Antibiotic
Chemical substance produced by a microorganism that has the capability of killing of inhibiting the growth of another organism
Antimicrobial agent
Range of activity of an antimicrobial agent against certain groups of bacteria
Spectrum of activity
Difference b/w intrinsic and acquired resistance
- Intrinsic: all members of the species are resistant
- Acquired: not all members of the species are resistant
Examples of intrinsic resistance
Staphylococcus saprophyticus and novobiocin
Examples of acquired resistance
Staphylococcus aureus and methicillin/oxacillin
Interpret susceptibility/resistance to oxacillin and cefoxitin as MSSA
MSSA is susceptible to oxacillin and cefoxitin
Interpret susceptibility/resistance to oxacillin and cefoxitin as MRSA
MRSA is resistant to oxacillin and cefoxitin
Three mechanisms used by bacteria in order to exchange genetic material resulting in antimicrobial resistance
- Genes
- Transfer
- Expression
Three mechanisms of bacterial resistance
- Impermeability
- Alterations in target molecules
- Enzymatic inactivation
Two mechanisms of impermeability
- Altered outer membrane porins
- Altered transport systems
Three mechanisms in altering the target molecules
- Methylation of rRNA
- Alterations of ribosomes
- Altered penicillin binding proteins
Three mechanisms in enzymatic inactivation
- Beta-lactamases
- Chloramphenicol acetyltransferases
- Aminoglycosides modifying enzymes
Three effects of combining antimicrobials
- Autonomous/indifferent
- Antagonistic (a substance that interferes w/ or inhibits the physiological action of another)
- Synergistic (one drug increases the other’s effectiveness)
What organism is universally susceptible to penicillin?
Streptococcus pyogenes
Standardizing susceptibility testing
- Growth medium
Mueller-Hinton Agar or broth
- Depth 3-5mm
Standardizing susceptibility testing
- pH
7.2-7.4
Standardizing susceptibility testing
- Cation concentration
- Mg2+
- Ca2+
- NaCl
Standardizing susceptibility testing
- Incubation conditions
35°C, ambient air, duration varies
Standardizing susceptibility testing
- Inoculum density
If not enough organism then not enough enzyme which could lead to a false susceptibility
Standardizing susceptibility testing
- Inoculum prep
3-5 colonies made into a McFarland standard
In a disk diffusion test (or an E-test), the agar depth is 1mm, what will the result be?
The antibiotic diffuses farther due to less agar → false susceptibility
In a disk diffusion test (or an E-test), the agar depth is 6 mm, what will the result be?
The antibiotic cannot diffuse as far → false resistance
In a disk diffusion test (or an E-test), a 0.25 McFarland is used, what will the result be?
There is less bacteria in the inoculum that is spread over the plate → Larger zone → false susceptibility
In a disk diffusion test (or an E-test), a 2.0 McFarland is used, what will the result be?
There is more bacteria in the inoculum that is spread over the plate → smaller zone → false resistance
In a broth dilution, a 0.25 McFarland is used, what will the result be?
There is less bacteria in the inoculum → ↓ MIC
In a broth dilution, a 2.0 McFarland is used, what will the result be?
There is more bacteria in the inoculum → ↑ MIC
Disk diffusion test
- What is the McFarland standard?
Uses barium sulfate to make a 1.5x10^8 CFU/mL standard which is a 0.5 McFarland
Disk diffusion test
- Inoculation of agar
W/in 15 minutes of prep, streak plates in 3 planes so that there is a lawn of confluent growth
Disk diffusion test
- Application of disks
W/in 15 minutes of inoculation, NEVER relocate a disk
Disk diffusion test
- Incubation
- 16-18 hours (Staph and oxacillin/methicillin for 24 hours)
- 35°C ambient air
Disk diffusion test
- Reading results
Read circular zone side in mm;
- If it’s resistant, zone size is still 6mm! NEVER 0!
Disk diffusion test
- Qualitative or quantitative?
- Interpretation of results
Qualitative → inform MD if its S, I, or R using CLSI charts
E-test
- What is the McFarland standard?
Uses barium sulfate to make a 1.5x10^8 CFU/mL standard which is a 0.5 McFarland
E-test
- Inoculation of agar
W/in 15 minutes of prep, streak plates in 3 planes so that there is a lawn of confluent growth
E-test
- Application of gradient strips
?
E-test
- Incubation
- 16-18 hours
- 35°C ambient air
E-test
- Reading results
Read elliptical zone side in mcg/mL
E-test
- MIC
Where growth intersects strip
- No zone: MIC > highest concentration
- Zone below strip: MIC < lowest concentration
E-test
- Qualitative or quantitative?
- Interpretation of results
Quantitative → informing MD of concentration (mcg/mL) and if its S, I, or R using CLSI tables
Broth dilution
- Inoculum prep
3.5 colonies and make a McFarland standard
Broth dilution
- What is the McFarland standard?
Use barium sulfate to make a 1.5x10^8 CFU/mL standard which is a 0.5 McFarland
- Final concentration: 5x10^5 CFU/mL
Broth dilution Macrotube
- Inoculation procedure
??
Broth dilution microtiter tray
- Inoculation procedure
- Deliver inoculum, via delivery prongs, into sterility tray with sterility well (neg) and growth control well (pos)
- Make a PURITY PLATE!
Broth dilution (Macrotube and microtiter tray) - Incubation
35°C ambient air, 16-20 hours
Broth dilution
- Reading results
MIC is the lowest concentration that inhibits growth
- Needs to be reproducible w/ one well margin of error
Broth dilution
- Qualitative or quantitative
Quantitative → informing MD of concentration (mcg/mL) and if it’s S, I, or R using CLSI tables
What is the significance of a regressive curve?
Linear and inversely proportional
- As zone size ↑, MIC ↓
Regressive curve
- X axis
- Y axis
- Y axis: MIC (mcg/mL)
- X axis: Zone size (mm)
- Top left = resistant
- Bottom right = susceptible
MIC/MBC procedure
Take clear tubes from MIC and incubate again on a plate. If < 50 colonies can grow on a plate thats considered killed!
Minimal bactericidal concentration
Killing capacity of 99.9% of original inoculum
MID/MBD procedure
?
MID procedure
Patient’s serum is serially diluted and a standard inoculum (5x10^5 CFU/ml) of patient organism is added. Incubate and read highest dilution which inhibits growth.
MBD procedure
- Highest dilution of patient serum that kills 99.9% of an original inoculum of patients organism
- Must be performed w/ MID
- Results are dilutions 1:8 to 1:32 is adequate
SXT is read at what % inhibition?
80%
Automated methods: Dade Microscan
- Three methods
- Traditional MIC methods
- Photometric reading (turbidity)
- Fluorometric reading (best method) (degradation of substrates by viable bacteria; detects bacterial inhibition by antibiotics)
Automated methods: BD Phoenix
- Three methods
- Gravity based inoculation process
- Redox indicator system
- Data management system
Automated methods: Vitek
- Three methods
- Computer-assisted analysis of growth
- Algorithm derived MIC
- Calculated MIC
Three methods of beta-lactamase detection
- Chromogenic cephalosporin: cefinase, nitrocefin
- Acidometric
- Iodometric
Results of beta-lactamase test
- Positive = organism produces enzyme to degrade penicilin, ampicillin, and amoxicillin
- Negative = does not produce enzyme but it may have some other mechanism of resistance to the same drug
What organism requires beta-lactamase testing?
Haemophilus influenzae
Which antibiotics which will not be effective against beta-lactamase producers
- Penicillin, ampicillin, amoxicillin
- Cephalosporins
- Carbapenems
What is the value of measuring serum levels?
Want to know how much antibiotic is at site of infection so that therapeutic range can be determined
Agar dilution
- Prep
Antibiotic is diluted into agar in 2-fold concentrations
Agar dilution is used for what type of organisms?
Fastidious organisms (N. gonorrhoeae, N. meningitidis)
Agar dilution, steers replicator
- What is the standard inoculum per spot?
10^4 CFU/mL
Agar dilution, steers replicator
- Incubation requirements
35°C, ambient air, 18-20 hours
What is the reference method for susceptibility testing?
Agar dilution
Five mechanisms of action a drug can have
- Inhibit cell wall synthesis
- Inhibit protein synthesis
- Inhibit nucleic acid synthesis
- Antimetabolites
- Alteration of cell membranes
What drugs work by inhibiting cell wall synthesis?
- Penicillins
- Cephalosporins
- Glycopeptides (inhibitis peptidoglycan synthesis; precursors of cell wall synthesis)
What drugs work by inhibiting protein synthesis?
- Aminoglycosides
- Tetracyclines
- Macrolides
- Clindamycin
What drug work by inhibiting the folic acid pathway?
Antimetabolites
What drug works by inhibiting DNA synthesis?
Fluoroquinolones
What drug alters cell membranes?
Bactracin
Mechanism of resistance
- DRSP
- Altered target sight
- PBP-2 pencillin binding protein
Mechanism of resistance
- ESBL
- Arise from point mutations of common beta-lactamases
- TEM-1 and SHV-1 genes coding for enzyme
Mechanism of resistance
- VRSA/GRSA
- May involve alterations in the cell wall and hyperexpression of PBP
- vanA gene present
Mechanism of resistance
- MRSA
- Alteration of target site (classic and modified)
- Hyperproduction of beta-lactamases
Mechanism of resistance
- VRE
- Altered peptidoglycan synthesis
- Carried by transposons or plasmids
Mechanism of resistance
- ARHI
Plasmid-mediated beta-lactamases (most common)
Mechanism of resistance
- CRE
- Carbapenemase (breaks down antibiotic)
- Cephalosporinase combined with porin loss
Lab detection of MRSA
- Cefoxitin disk diffusion
Induces mecA gene (best drug to detect MRSA)
- Media: CAMHB with 2% NaCl
- Incubation: 24 Hours, 35°C, ambient air
Lab detection of DRSP
- Disk diffusion w/ oxacillin
Disk Diffusion using Oxacillin;
- Media: Mueller-Hinton Agar, 5% SBA;
- Incubation: 20-24 Hrs, 35°C in CO2;
Lab detection of ARHI
- Disk diffusion
- Media: HTM agar
- Incubation: 16-18 Hrs, 35°C in CO2
Lab detection of VRE
- Disk diffusion
- Media: Mueller-Hinton Agar;
- Incubation: 24 Hrs, 35’C, Ambient Air;
Lab detection of VRSA/GRSA
Disk Diffusion and Broth Dilution tests are held for 24 Hrs;
Agar Screen:
- Media: BHI with 6ug/mL Vancomycin,
- Incubation: 24 Hrs, 35’C, ambient air;
D-Test Disk Induction: Erythromycin and Clindamycin disks are placed 15mm apart and flattening is noted; Pos=Resistant to clindamycin, Neg=susceptible
Lab detection of ESBL
Confirmatory Test: Use cefotaxime and ceftazidime disks and each disk combined with clavulanic acid; >5mm increase with clav=pos;
Can also use E-Test Strips, Vitek-2, and Microscan
Lab detection of CRE
Modified Hodge Test: 1:10 Dilution of 0.5 Mcfarland (E.coli lawn)(because larger zone size with decreased inoculum), streaked with known + and - and Patient.
- Shouldering occurs if enzyme present to break down meropenam = pos result/resistance
MRSA
- Infection types
- HA-MRSA
- CA-MRSA
What is HA-MRSA?
- Found in ICUs
- S. aureus has acquired several genes that are responsible for its resistance
What is CA-MRSA?
- S. aureus has only gene responsible for its resistance
VRE
- Infection types
- Nosocomial bacteremia
- Surgical wound infection
- UTI
DRSP
- Infection types
- Otitis media
- Sinusitis
- Community acquired pneumonia
- Bacteremia
- Meningitis
ARHI
- Infection types
?
VISA/VRSA
- Infection types
?
ESBL
- Infection types
- E. coli
- Klebseilla spp
- Proteus mirabilis
CRE
- Infection types
- Class A: K. pneumoniae
- Class B: P. aeruginosa, A. baumannii
- Class D: A. baumannii
Effective treatment or prevention
- MRSA
- Linezolid
- Synercid
- Daptomycin
- Vancomycin to treat
Effective treatment or prevention
- ARHI
- Type b vaccine
- Empiric treatment w/ cefotaxime or ceftriaxone
Effective treatment or prevention
- DRSP??
??
Effective treatment or prevention
- VRE??
??
Effective treatment or prevention
- VRSA/VISA??
??
Effective treatment or prevention
- ESBL??
??
Effective treatment or prevention
- CREs??
??
Synercid treats which problematic bacteria?
- MRSA
- VRE
Daptomycin treats which problematic bacteria?
- MRSA
- VRE
Telithromycin treats which problematic organisms?
Macrolide-resistant pneumococci
What test detects the inducible clindamycin resistance mechanism in GP organisms?
D-test
Desribe the prinicple behind the D-test
Erythromycin is used to induce the resistance mechanism (ribosome alteration) to clindamycin w/in the GP organism
- If using the D-test, the GP organism should already be resistant to erythromycin
Describe the procedure of the D-test
Erythromycin and clindamycin disks are placed 15mm apart and flattening is noted around the clindamycin disk
Interpret the results of the D-test
- Pos: GP organism is RESISTANT to clindamycin (CANNOT USE FOR TREATMENT!)
- Neg: GP organism is SUSCEPTIBLE to clindamycin
Rationale for identifying VRE to the species level
To confirm if we truly have VRE (E. casseliflavus or E. gallinarium)
Resistance mechanism responsible for vancomycin resistance in S. aureus (VRSA)
Alteration of cell wall
Resistance mechanism responsible for penicillin resistance in S. pneumoniae
Alteration of target site
What antibiotic disk is used to predict penicillin resistance in S. pneumoniae?
Oxacillin
What antibiotic disk is used to predict oxacillin resistance in S. aureus?
Cefoxitin
What is the purpose of the confirmatory ESBL test?
To detect the presence of extended spectrum beta-lactamase enzymes produced by an isolate
Four disks used in the confirmatory ESBL test
- Cephalosporin (CTX)
- Clavulanic acid (CLA)
- Cephalosporin/clavulanic acid (CTX/CLA)
- Ceftazidime/clavulanic acid (CAZ/CLA)
What is the purpose of the cefoxitin screen?
To determine if an isolate produces the mecA gene (differentiates MSSA vs. MRSA)
What size zone indicates a “positive” (resistant) screen?
≤ 21mm (MRSA)
What size zone indicates a “negative” (susceptible) screen?
≥ 22mm (MSSA)
CRE/modified Hodge test
- Purpose
To detect the presence of carbapenemases
CRE/modified Hodge test
- What organism is streaked all over the plate?
E. coli
CRE/modified Hodge test
What disk is placed in the center of the plate?
Meropenem
What does “MRSA” stand for?
Methicillin resistant Staphylococcus aureus
What does “DRSP” stand for?
Drug resistant Streptococcus pneumoniae
What does “ARHI” stand for?
Ampicillin resistant Haemophilus influenzae
What does “VRE” stand for?
Vancomycin resistant Enterococcus spp
What does VRSA/VISA” stand for?
Vancomycin resistant Staphylococcus aureus
What does “ESBL” stand for?
Extended spectrumm beta-lactamases
What does “CRE” stand for?
Carbapenem resistant enterics
Lab detection of MRSA
- MRSA agar screen
Meuller-Hinton Agar + 4% NaCl + 6 mcg/mL Oxacillin
Lab detection of DRSP
- Broth dilution
- Media CAMHB with 2-5% LHB;
- Incubation: 20-24 Hrs at 35°C, ambient air
Lab detection of DRSP
- E-test
- Media: Mueller-Hinton agar, 5% SBA;
- Incubation: 20-24 Hours 35°C in CO2
Lab detection of ARHI
- Broth dilution
- Media: HTM broth
- Incubation: 20-24 Hours, 35°C, ambient air
Lab detection ARHI
- Nitrocefin assay
Chromogenic cephalosporin
- Pos: isolate is resistant to penicillin, ampicillin, and amoxicillin
- Neg: isolate can be resistant by other mechanisms
Lab detection of VRE
- Broth dilution
Broth Dilution:
- Media: CAMHB;
- Incubation: 24 Hrs, 35’C, ambient air; Agar
Lab detection
- Screening test
- Media: BHI with 6ug/mL vacomycin;
- Incubation: 24 Hrs, 35’C, Ambient air, any growth = resistance;
Confirmation of VRE with gram stain
