from karen's review ppt
list the three ways to ID bacteria (general)
biochemical
proteomic (maldi)
molecular
biochemical identification of bacteria
automated: vitek 2, phoenix, microscan
bench top
molecular identification of bacteria
hybridization probes
targeted naat
multiplex pcr
16S pcr/sequencing
what is maldi tof?
matrix assisted laser desorption/ionization time of flight
matrix, organism, slide, laser
add formic acid, need isolated colonies
list the components involved in maldi tof
ionization chamber, laser beam, mass spec analyzer, ion detector
maldi tof theory
bacterial colony is zapped with a laser
ribosomal protein fragments are separated by size
peaks are compared to database = ID
maldi tof advantages
quick ID in 1 hr
low daily costs
easy to use and update databases
expanding applications (molds, AFB, AST)
maldi tof disadvantages
high initial cost
maintenance
limited by database
media selection
organism grown in cx vs direct specimen
age of organism
growth of organism
what is hybridization?
formation of hydrogen bonds b/w dna/rna strands complementary to each other
probe binds with target
what is a target?
the nucleic acid sequence we want to identify
is it in our sample?
what is a probe?
designed based on target sequence and tagged for detection
detected via radionucleotides, enzymatic labeling, fluoro
advantages of hybridization
easy to use
quick
rapid ID of pathogen in cx
blood = fluoro
AFB, fungi, bacteria = fluoro via probe and hybridization
can be used on pt specimen
disadvantages of hybridization
only works well directly on select specimens/bugs and or clinical situations
low analytical sensitivity
list the PCR reagents
dna pol, buffer, primers, dNTPs, template, thermalcycler
dna pol
enzyme that couples nucleotides to the dna chain
buffer
maintains proper pH for rxn to take place (MgCl2)
primers
small nucleotide chains that are complementary to the sequence we wish to amplify
dNTPs
nucleotides used to elongate the duplicated dna strand
template
the original dna strand we wish to duplicate
thermalcycler
a variable temperature block to control the rxns taking place in the 96 well plate
PCR steps
denaturation, annealing, extension, repeat 30-40 times
denaturation
disrupt H bonds (double helix falls apart)
95C
annealing
primer binds to target sequence
starting point for dna pol to make new sequence
temp decreased: dependent on the temp for specific primer used
extension/elongation
primer extension by dna pol by adding nucleotides
gel electrophoresis
used to detect if PCR amplification was successful
takes long time to run and requires system to view gels
generates hazardous waste
real time PCR
look for product at each cycle end for exponential signal related to amplified genetic material
detection:
specific: hybridization probe binds target
non specific: sybr green binds dsDNA
qualitative: set a certain cycle number as binary threshold
quantitative: calibrate cycle # to standard curve so we get estimate of target template amt
advantages of real time PCR
no waiting, results known as rxn takes place
use pt samples
rxns happen in closed tube = dec contam
imaging system part of it
disadvantages of real time pcr
relative quantitation
log scale
lack of standardization
detects very small amts of genetic material
so is it a true infection?
multiplex PCR
screens for multiple targets at once
resp: viruses, bacteria
blood cx: GN, GP, yeast, resistance
meningitis: bacteria, viruses, yeast
GI: bacteria, e coli/shig, viruses, parasites
advantages of multiplex PCR
tests for many pathogens all at once
rapid TAT
little hands on processing
disadvantages of multiplex PCR
costly
impact to pt care unknown
interpretation of specimens with multiple positives
ribosomal sequencing
needs to grow in cx
easy to ID isolate
broad range ribosomal sequencing
performed directly on tissue/BF
conserved gene sequencing
16s RNA = bacteria
18s RNA = yeast
harvest dna from cx
amplify with PCR, send for sanger, search database
sanger sequencing
ddNTPs added randomly in PCR, stops elongation
missing OH- group
capillary electrophoresis: fluoro tagged ddNTPs emit light as they move thru tube = sequence
compare to database
single amplicon from single sample is amplified and single sequence is obtained
NGS
high throughput, low cost
add a base, detect signal (if bound), cleave dye, add another base
detect each time base is added in real time
sequencing millions of fragments simultaneously
NAATs
quick ID of important organisms in cx OR directly from specimens
MRSA in cx/skin swabs
MTB in cx/sputum
c. diff in stool
viral loads in blood/BFs
zone of inhibition
diameter of absence of bacterial growth around disk indicates S, I, R
MIC - minimum inhibitory conc
the lowest conc of an antimicrobial that inhibits growth
MBC - minimum bactericidal conc
the lowest conc of an antimicrobial that kills the organism
synergism
combination of antimicrobials provides inc activity than the sum of effects of agents separately
treatment for enterococci
penicillin and aminoglycosides
empirical treatment
based on standard susceptibility of an organism (ex: beta strep, penicillin)
inducible resistance
resistant only see when induced by exposure to another agent (ex: clindamycin, erythromycin = D test)
intrinsic resistance
the organism is inherently resistant to an antimicrobial
bactericidal
kills bacteria
bacteriostatic
inhibits bacterial growth w/o killing (ex: STX)
what should you give if pt is allergic to penicillin?
erythromycin
side effect of chloramphenicol
aplastic anemia
side effect of aminoglycosides
renal and ototoxicity
side effect of tetracycline
browns teeth (don’t give to kids)
what drugs inhibit cell wall synthesis?
penicillins, cephalos, vanc
what drugs inhibit protein synthesis?
aminoglycosides, tetracyclines
what drugs inhibit folic acid synthesis?
sulfonamides
chloramphenicol mode of action
prevents mRNA from attaching to ribosomes for protein synthesis
what drugs inhibit dna replication?
quinolones
what is h. influenzae susceptible to?
ampicillin
what is m. cat usually resistant to?
penicillin, ampicillin (beta lactamase producer)
what is klebsiella usually resistant to?
ampicillin and carbencillin
what is pseudomonas usually resistant to?
many drug (STX)
what is steno usually resistant to?
many drugs (S to STX)
antibiogram
aids physician while they wait for susceptibility report
confirms ID of organism based on AST
drug only given in a urine culture?
nitrofurantoin
proteus
R to tetracycline and nitrofurantoin
kirby bauer
determines S, I, R based on ZOI
0.5 McF (barium sulfate is std)
usually MH, but can change based on organism
read within 16-18 hrs (except vanc testing = 24 hrs)
what can cause the ZOI to look too susceptible?
thin agar, delayed incubation, pH, low temp, disk too potent
(anything that gives drug head start)
what can cause the ZOI to look too resistant?
agar dried out, pH, heavy inoculum, disk not tamped, delayed disk placement, outdated disk, long incubation
(anything that gives organism head start)
e test
ellipse is intersection of strip and MIC
MIC is given which determines S, I, R
inter colonies
on e test and KB
need to sub them and retest the susceptibility
most likely more resistant so need to report the more resistance MIC
broth tube dilution
gold standard
MIC is the first tube that has no growth
need control without any antimicrobial
schlicter
determination of the lowest dilution of pt’s serum with drug that kills that organism
serially dilute pt serum and add organism to suspension
MIC is last tube w no visible growth
beta lactamase test - cefinase - nitrocefin
pos color is red
how to we test for MRSA?
oxacillin screening agar with NaCl OR oxacillin/cefoxitin disks
how to we test for VRE?
screening agar with vanc for 24 hrs
what organisms are usually VRE?
e. faecalis, e. faecium
what organisms are usually VIE?
e. casseflavus, gallinarum