review flashcards

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