Antibiotic Susceptibility Testing Flashcards
meant to kill off any invading organism
bactericidal
-used in immunocompromised patients
-life threatening
prevents further growth of the organism
bacteriostatic
-healthy people
-non life threatening diseases
lower dosage
bacteriostatic
higher dosage
bactericidal
broad spectrum drugs
activity against wide range of organisms
-good when we don’t know the organism
downfall= kill more normal flora
narrow spectrum antibiotics
limited to target range
best if pathogen known
synergy means
2 drugs when given together work better together than on their own
antagonism means
if I give you one drug it will counteract another drug
is there an ideal antibiotic?
no there is not an antibiotic that fits all
look at cost
site of infection
how long from inception to market does a new drug take
over 10 years
-need volunteers to test safe
-resistance happens fast
Average time from hitting market to resistance
6 months
most resistance organisms
e.coli
superbug- show multi resistance
MDR= multiple drug resistance
organism is resistant to 1 agent in 3 or more antibiotic classes
XDR= extra drug resistant
resistant in one agent in all but 2 or fewer antibiotic classes
PAN= pan drug resistant
resistant to everything
- some e.coli and pseudomonas
-treat with cocktail of drugs
MRSA
methicillin resistant staph aureus
VRE
vancomycin resistant enterococci
strep pneumo is resistant to
penicillin
why so much resistance?
antibiotics in agricultural
not finishing antibiotics
laundry detergent, hand sanitizer
what is intrinsic resistance?
bacteria is naturally resistant
this is a way we ID some organisms
how does acquired resistance occur?
target site modification
plasmids
efflux
enzymes
jumping genes
target site modification
target certain site of bacteria and the bacteria changes the site
plasmids
extra chromosomal piece of DNA
-resistance gene is found here
-easily transferable from one bacteria to another
efflux
antibiotic goes in and organism spits it out
jumping genes
transpanozone????
jumps from pieces of DNA to another
things to think about with antibiotic selection
resistance, cost, patient population, dosages, location
primary drug
first line that has been manufactured in the category
§ From nature, ex. Penicillin
§ Cheaper
§ Less toxic to human
Good treating
secondary drug
chemical modifications from primary drugs
-due to resistance of primary drug
tertiary drug
alter side chain
farther down the line in modifcations of drugs=
higher cost of new drug
more side effects seen in human
treat more poly microbal infections
advantage of more modifications to drugs
treat more resistant organisms we are now seeing
standard for inoculum
Mc Farland Standards
-sets turbidity standards that allow us to determine the amount of organism present in a broth
-we buy
how to create turbidity
barium chloride
sulfuric acid
-now it is given in latex particles
higher you go with Mc Farland standards =
more turbidity = more organisms in your broth if matching turbidity standard
most common amount of standard
0.5 Mc Farland Standard = 1.5 x 10 ^8 CFU /mL
what is MIC
Concentration of antibiotic in mg/mL that prevents the in vitro growth of bacteria
minimum
how is MIC done
○ 1 control= broth and organism - makes sure organism is viable ; growth
○ Broth control= Mueller Hinton: make sure broth isn’t contaminated ; no growth
○ Antibiotic control= antibiotic and broth= make sure antibiotic isn’t contaminated ; no growth
○ Incubated 35 degrees 18-35 hours
MBC
Minimum bactericidal concentration of drug
info about MBC
tubes show no turbidity
plated on BAP
look for CFU on actual plate
1st plate that shows 99% reduction of organism= MBC
-higher concentration than MIC
bacteriostatic drugs
kirby bauer principle
zone of inhibition is created as a result of antibiotics diffusing away from the disk
read in millimeters
why can’t let bacteria sit more than 15 minutes before plating on Mueller Hinton plate
bacteria will double
-almost immediately antibiotic will start working so can’t move disk
method of detection set by
CLSI- clinical laboratory standards institute
Muller-Hinton measurements
150 millimeters across
depth 4 millimeters
if depth on muller hinton is greater
we will get false resistance
if depth is less than normal on a muller hinton plate
false susceptibility
how many disks placed on muller hinton
12 disks
24 mm apart
pH on muller hinton
7.2-7.3 at RT
too low pH on muller hinton=
false resistant with amino glycosides
too high pH on muller hinton plate
false susceptibility with amino glycosides
what is muller hinton grown in
ambient air (O2) not CO2 but depends on organism
CO2 will decrease pH
decrease temp when growing muller hinton
false susceptibility
bigger zone sites
only time a muller hinton plate can grow at 35 degrees
MRSA
can be incubated at 30, Mec A expressed better at 30 and full 24 hrs
concentrations of what are importatnt in Muller-Honton when testing amino glycosides
calcium, magnesium and zinc
when are amino glycoside used
for pseudomonas
if concentration is increased =
false resistance
if concentration decreased=
false susceptibility
drug concentrations -each disk of antibiotics concentration set by
FDA
how to store disks
refridgerator for up to a week
if not used in a week, store in a frost free freezer at -20
-frost will cause moisture and antibiotics will leak out
before reporting any results we need to do
QC on disks and representative organisms
organisms are ATCC
what organisms used for QC
○ Always use an E.coli, S. aureus, and pseudomonas
E.coli- gram- , s. aureus, gram +, pseudomonas, non fermenter
when first starting antibiotic panel have to do QC
everyday for a month
- If during the 1 month panel and have less than 3 antibiotics outside accepted readings then can do QC weekly
how read zones on regular muller plate
read from back to plate
edge to edge
read above dark surface from back site with reflective light
how to read muller hinton with added choc or blood
read from top side with top off
if there is more than 1 organism present on a plate
plate to a purity plate
proteus ignore swarming and read outer edges
sulfa- read best defined edge
if 2 or 3 colonies in the zone
can’t report out
-purity plate
-hetero resistance -seen in MRSA
what is hetero resistance
§ Not every colony in a population expressed resistance to the same degree
§ If see 1 colony in the zone, need to consider entire drug to be resistant
SIR method of reporting based on
CLSI
sensitive
sensitive to standard dose of drug that is used for the antibiotic
intermediate
some things might cause drug to be resistant in vivo
Not first choice of drugs
resistant
don’t use this drug
how to determine break points?
regression analysis
SIR are breakpoints
○ Test hundreds of different organisms using standard dose that should go on each disk
○ Plot MIC against zone size = determine where we should establish the break points
how to do a manual muller-hinton
cut plate in half and lawn upper half, rotate and repeat 2 more times
rim edges
e testing known as
gradient disk method
elliptical pattern
advantage of E testing
we can get MIC
used in life threatening organisms
disadvantage of E testing
can’t test 12 antibiotics
expensive to do
where the drug intercepts the drug pattern-
MIC amount of drug can be used
screen for beta lactamase enzyme which can break down the ring
cefainse disk
has nitrocefin on it
resistant means can’t use beta lactam drugs
-can occur in multiple different ways
antibiotic on _____ disk
nitrocefin
groups of drugs known as sulfasporein
only time we do the disk is when
the organism is capable of producing enzymes
ex. H.flu, moraxella, neisseria gonorrhea
only test we do on H. influenza
beta lactamase
moraxella
used to be 100% penicillin sensitive
now 98% penicillin resistant
neisseria gonorrhea
some places do it with beta strep group A
-beta strep group A considered universally susceptible to penicillin
no beta lactamase on
MRSA
-confirm through a different method
majority of times beta lactam drugs inhibit
cell wall synthase of organism
-sites where enzymes are referred to penicillin binding proteins (PBP)
when drug binds to protein stops the cell wall synthase
3 modes of resistance
-production of beta lactamase
-altered binding protein sites
-efflux
first method used to screen for MRSA
Oxacillin
could see heteroresistance
instead of using oxacillin we want to now test with
cefoxitin
-helps mec a be better expressed
1st step in finding MRSA
resistant to cefoxitin
once MRSA is ID as having resistance to cefoxitin and oxacillin what is done
PBP2a latex test
recommended for detecting MRSA
1- Recommended that do not read susceptibility testing for a full 24 hours
2- Tested at 30 degrees
3-Add 2-4% sodium chloride to all plates used for MRSA testing
resistance with cefoxitin means
no penicillin drugs can be used
after testing with cefoxitin it gets sent to
PCR to look for Mec A gene
1 drawback with testing with cefoxitin
not accurate results with spinal fluid
Spinal fluid should be sent for PCR testing
PCR can not look for presence of
Mec C gene
if organism is not a MRSA can still use
penicillin, methicillin, oxacillin
screening tool for MRSA
chromogenic agar
-typically cefoxitin with color changing substrate
-DO NOT use colonies on this agar for susceptibility testing
most resistance to vanco is because
altered binding sites
some have beta lactamase that can confer resistance to vanco
glycopeptide
VISA
vancomycin intermediate S.aureus
4-8
retest before reporting out
VRSA
vancomycin resistant s.aureus
> 16 ug/mL
CDC reportable
must retest before reporting out
beta lactamase inhibitor
Group of drugs similar to beta lactam drug and bind to a beta lactamase enzyme to stop the action of the enzyme
bactericidal actions as well
examples of beta lactamase inhibitor
- Clavulanic acid
- Sulbactam
- Tazobactam
- Avibactam
- Ampicillin/ sulbactam
- Ticarcillin/ clavulanic acid
- Piperacillin/ tazobactam
DONT work with MRSA
know if beta lactamase inhibitors work
- Zone size >5mm from original zone size, the beta lactamase inhibitor will work
inducible resistance
to clindamycin
gram + cocci
if organism has been exposed to erythromycin this can cause it to be resistant to clindamycin
if resistant from onset
no inducible resistance
NO D test
if resistant to erythromycin and suspectible to clindamycin MUST
perform D test
flatten side of D pointed toward erythro.
what gene causes clindamycin inducible resistance
erm gene and NOW msr gene
place antibiotics 15-20 mm apart
D+= resistant to both
done on S.aureus or CNS
VRE
vancomycin resistant enterococci
VRE genes resistant
- Van A, Van B, Van C, Van E, Van G
○ Most common A and B
confer via a plasmid
van c
intrinsic resistance, we don’t need to worry about
○ E.gallinarium
○ E.flavescens
○ E.casseliflavens
most common VRE seen in lab
- E.faecium
- E.faecalis
- Don’t have to be VRE, just most common to be
VRE requires patient to be isolated in a hospital because
lives in gut and if have stool on hand can be easily transferred patient to patient
how to detect VRE?
vanco plates good screens
6mg of vanco on ??
if organism grows= vanco resistant
done for epidemiology purposes
gentamicin belongs to
amino glycosides
enterococci are intrinsically resistant to
small amounts of amino glycosides
some enterococci can be treated with
combing a cell wall agent with aminoglycoside
synergistic affect
used on VRE
Now some enterococci have plasmids that are conferring resistance
to high levels of amino glycosides
now synergistic relationship can’t be used
if resistance to gentamicin
500 mg/mL considered high level resistance and can’t use synergistic drugs
esbl
extended spectrum beta lactamase
-gram - organisms
resistance goes to cephalosporins
first 2 noted ESBLs
klebsiella and e.coli
-now go into enterobacterace
if organism is reported ESBL need to report R to
cephalosporins, aztreonam, and all penicillin
different enzymes that cause ESBL
- TEM-1
- SHV-1
- CTX-M
- OXA
how to determine if ESBL
indicator drugs
ceftazidime and cefotaxime
if resistance to any one of indicator drugs = pair with beta lactamase indicator
if increase of at least 5 mm or more in zone size=
ESBL
-after confirmed need to use carbapenems
SPICE (space) organisms INDUCED resistance to
Amp C gene
chromosomal beta-lactamase genes
gram -
most organisms have AMP C gene that is not expressed
if organism has a not expressed Amp C gene and come in contact with ______ gene will be expressed
cephalosporins
this is due to Amp C gene
enzymes known as _________ that confer resistance to carbapenem drugs
carbapenmases
-plasmid mediated enzymes
known as KPC or CRE
KPC
klebsiella pneumonia carbapenmases
CRE
carbapenem resistance enterobactace
now
CRE resistant to
- Penicillin’s
- Cephalosporins
- Aztreonam
- Carbapenems
if you have a CRE what drug is used to treat
tigecycline
polymyxin B -was off the list because neprho toxin- can’t use on kidney patient
how to determine CRE
look for indicator drugs
○ Imipenem
○ Meropenem
○ Ertapenem - majority use this
what CRE indicator drug is best
ertapenem
better results than other 2, express carbapenmase enzyme
main class of beta lactamase enzyme
class b= metallo-B-lactamase
metallo-B-lactamase enzyme requires
metal ion to hydrolyze beta lactam ring
metal used: magnesium and zinc
New metallo beta lactamase enzyme found
new delhi- class B
not inhibited by beta lactamase inhibitors
inhibited by EDTA
test for carbpenmases
Detect presence of class A,B,C,D but doesn’t say which class it is
do this test if ID as CRE
look for chromogenic changes
inhibitors of cell wall synthesis
mode of action
don’t allow cell walls to be made by bacteria
majority of beta lactam drugs are
bactericidal
oldest group of antibiotics
side chains easily manipulated
efficacy against gram - and +
natural penicillin
○ Penicillin G- given shot
○ Penicillin V- oral
semi-synthetic penicillin
○ Methicillin
○ Oxacillin
○ Nafcillin
○ Ampicillin
Amoxicillin
newest generation of penicillin
piperacillin
where do cephalosporins come from
acremonium
-contain a beta lactam ring
what generation of cephalosporins can cross the blood brain barrier
3rd generation
up to 5th now
cephalosporins better at what gram
positive
cephalosporins inhibitor of
cell wall synthesis
monobactams only used for
gram - organisms
inhibitor of cell wall synthesis
includes aztreonam
carbapenems are inhibitors of
cell wall synthesis
ertapenem
Meropenem
Imipenem
carbapenems efficacy against
gram + and gram -
DOES not work against VRE or MRSA
glycopeptides inhibitors of
cell wall
main glycopeptides
vancomycin
no beta lactam ring
nephro toxic
only gram +
IV best way to administer
glycopeptides can’t penetrate
cell wall of gram - organisms
how does vanco resistance happen
efflux technique
choice of treatment for MRSA
vancomycin
inhibitors of cell membrane synthesis are more toxic to
humans
disrupt formation of cell membrane or alter membrane and things leak out
topical agents typically
bacitracin
topical agent
ID beta strep group A
polymyxin
topical agent
taken off the market because extremely nephro toxic
now back
how does inhibitors of protein synthesis work
binding to 30S or 50S ribosomal units
used for gram + or -
aminoglycosides
inhibitor of protein synthesis
gentamycin -crosses most often
Kanamycin
Tobramycin
Amikacin
Streptomycin-secondary drug for myco
aminoglycosides mainly used on
gram -
less toxic against gram +
nephro and odo toxic
what values need to be run on people with aminoglycosides
bun and creatinine
monitor kidney
can’t use on pregnant women
tetracycline
binds calcium and cause discoloration of fetal and adult teeth- permanent
tetracycline new generation
tigecyclines
-last resort
what kind of organisms is tetracycline best for
intracellular
chlamydia
macrolides
inhibitor of protein synthesis
erhyomycin- if allergic to penicillin
azithromycin-strep Z pack
chloramphenicol- cause aplastic anemia
drug of choice for haemophilus influ
chloramphenicol
inhibitors of folic acid synthesis
sulfa drugs -work by inhibiting folic acid pathway
used for UTI
concentrate urinary tract
nucleic acid synthesis
inhibit enzymes needed for DNA or RNA synthesis
fluoroquinolones
against gram - and anaerobic
riframpin- used for TB
staph saprophyticus intrinsic resistance to
novobiocin
leuconostoc intrinsic resistance to
vancomycin
listeria intrinsic resistance to
ceftriaxone
enterococcus gallinarium intrinsic resistance to
vancomycin
proteus intrinsic resistance to
tetracycline
klebsiella intrinsic resistance to
ampicillin, ticarcillin and carbenicillin
serratia intrinsic resistance to
Ampicillin, erythromycin, and Cephalothin
always recheck before reporting out
MRSA, VISA, VRSA, beta strep group A, erythromycin R and clindamycin S
