antibiotic susceptibility testing and antimicrobial resistance 2

when is susceptibility testing needed?

when a bacterial isolate is determined to be a probable cause of the patient’s infection and the susceptiblity of isolate cannot be readily predicted.

when pt is allerigc to standard treatment regiment

in cases of antibiotic treatment failure

what is cascade reporting

when lab only reports certain antibioticcs results based on the suceptibility of first line drugs

ex. if bacteria is susceptible to narrow spectrum antibiotic lab may withhold reporting the board spectrum unless rquested to

why is cascade reporting important

prevents the overuse of broad spectrum antibiotics

reduce antibiotic resistance

what are antibiograms

used to compare susceptilbity patterns of specific bacterial isolates

• allows for gradual resistance patterns

• allows for guideline for treatment before susceptiblity testing is complete

what is susceptible

antibiotic will inhibit growth or kill the microorganism

what is resistance

antibiotic will not inhibit growth of the microorganism

what is intermediate

the antibiotic will likey work if higher/more frequent doses could be used

what is susceptible dose dependent

the antibiotic will only work with additional dose / higher dosage

what is the kriby bauer disk diffusion procedure

standardized amount of bacteria is set up as a lawn on a mueller hinton agar plate

paper disk that is impregnated with antibiotics are place onto the surface of agar

incubated for 18-24 hours and read

the mm of the zone in diameter is measured and interpreted

what causes a false negative KB disk diffusion

if the agar depth is deeper than 4mm

allows the amount of antibiotic to go further down in the plate and makes the organism appear more resistant

what causes a false positive KB disk diffusion

if the agar depth is thinner than 4mm

allow for more antibiotic to stay on the surface of agar and make the organism appear more susceptible than it is

what should media pH be for KB disk diffusion

7.2-7.4

what is tray dilution

allows for multiple dilution of a drug to be tested with the same organism

serial dilutions are used usually doubling dilution or two-fold dilutions

what does MIC stand for

minimal inhibitory concentration

how is tray dilution recorded

the smallest diltuion of antibiotic with no growth

what is the e-test

epsilometer test

allows for MIC determination in a “disk diffusion” like method

e-test procedure

a gradient strip containing various amounts of antibiotics

lawn of bacteria is streaked and the strip is laid on the lawn

incubate for 18-24 hours then read

what does the MIC equal to on a E-test

equals the number on the scale where the zone of inhibition touches the strip

why do automated methods do not work on anaerobic susceptibility testing

needs to stay in the anaerobic environment

what are other tests for anaerobic susceptibility testing

e-test and microbroth dilution trays

what agar should be used for e-test in anaerobic susceptibility testing

brucella agar with a 1.0 macfarland standard

incubate at 24 hours

AST of fastidious organism - streptococcus spp.

muller hinton agar supplement with 5% sheep blood

muller hinton broth supplemented with 2-5% lysed horse blood

incubate in 5% CO2 for 20-24 hours

AST of fastidious organism - haemophilus

haemophilus test medium (HTM) in broth or agar is required

AST of fastidious organism - anaerobes

brucella laked sheep blood, increase test inoculum incubate anaerobically for 48h

what are peak and through antibiotic levels

potentially toxin antibiotic must be monitored to determined amount in the blood

peak levels are

collected 30 minutes post IV antibiotic administration or 60 minutes post IM drug administration

what does peak show

shows the highest amount of the antibiotic in the patient’s blood

what is through levels

drawn right before the next dose of antibiotic to show the lowest levels of the drug

what does resistance mean

an organism that has a mechanism to keep that antibiotic from working

what are the 2 different types of resistances

intrinsic and acquired

why is differentaing between intrinsic and acquired resistance important

when determining apporpriate infection control isolation precaution

what is intrinsic resistance

chromosomally encoded, predictable resistance

natural resistance that the organism did not develop

“born with it”

intrinsic resistance example - enterobacterales and vancomycin

vanco is too big to get through the gram negative cell wall

intrinsic resistance example - staphylococcus saprophyticus and novobiocin

Gyrb gene point mutation that effects drug binding site

intrinsic resistance example - klebsiella spp. and ampicillin

SHV gene beta-lactamase degrade amipiclinan before reach penicillin binding protein

what is acquired resistance

shared from another organism or “learned” - requires previous exposure to the antibiotic

ogranism that was orginally susceptible to a drug when tested now considered resistant after the drug it was used to treat

acquired resistance mechanism mutation - transformation and recombination

uptake of naked donor DNA which combines with recipient homologous DNA

acquired resistance mechanism mutation - transduction

bacteriophages incorportated bacterial DNA into the viral genome upon replication and then can pass along to newly infected bacteria

acquired resistance mechanism mutation - conjugation

chromosomal DNA, plasmids or transponson are transferred between bacteria via pili

what are the types of resistance mechanis

production of enzymes

preventing uptake f the drug

modification of the drug binding site

inactivation of the drug

efflux

what are beta lactamase producers

makes enzymes that breaks down penicillin and 1st gen cephalosporins

what is MRSA

methicillin resistant staphylococcus aureus

what is VRE

vancomycin resistant enterococcus (e.faecalis)

what is ESBL

extended spectrum beta lactamse producer

what is CRO

carbampenem resistant organism (CRE, CRPA, CRA)

what is VRSA

cancomycin resistant staphylococcus aureus

what is the ntirocefin/cefinase test

is the cephalosproin commonly used to screen for beta lactamase production

filter paper contain nitrocefin

paper is wet then inoculated with small amount of organism to be tested

nitrocefin/cefinase principle

if an organism produces beta lactamase the bond of the beta lactam ring in the nitrocefin will hydrolyze causing color change from yellow to red

what does the nitrocefin/cefinase test predicts

penicillins, ampicllin, amoxicillin, varbenicllin, piperacillin results

what does nitrocefin/cefinase test detects

beta-lactamase production particcular for haemophilus, neisseria moxaxella staphylococcus enterococcus and anaerobes

what organism should nitrocefin/cefinase test not be used for

aerobic gram negative bacilli

what is MSSA

methicillin susceptible a.aureus

s.aureus strains that are susceptible to antistaphylococcal penicllins

what is MRSA resistant to

all beta lactam antibiotics including cephalosporins and carbapenems

Which antibiotic is currently used for testing MRSA?

Oxacillin or cefoxitin (methicillin is no longer used clinically)

MRSA is commonly acquired by

hospital infection

where do people are MRSA

in th nares

what gene is responisble for MRSA resistance?

mecA gene - encodes an altered penicillin binding protein (PBP2a)

What is the function of PBP2a encoded by mecA?

allows cell wall synthesis to continue enven in the presence of beta lactam antibiotics

Where is the mecA gene located in MRSA?

On SCCmec (staphylococcal cassette chromosome mec), a mobile genetic element that inserts at the same site on the S. aureus chromosome

MRSA testing methods - PCR for mecA (mecC) and SCCmec

looks for gene but not nessarily expresion

MRSA testing methods - cefoxitin disk screening test

cefoxintin is an inducer for the mecA gene

0.5 mcfarland of organism then lawn and cefoxitin as disk

S.aureus zone < 21 mm shows resistance

MRSA testing methods - oxacillin agar screen

NaCl supplemented mueller hinton agar that contain oxacillin

growth indicates resistance to oxaillin

MRSA testing methods - chromogenic agar

selective and differential media for MRSA

used in screening population but not for normal resistance testing

Which genes are associated with vancomycin resistance in Enterococci (VRE)?

vanA, vanB, vanC, vanD, vanE, vanG

what are common genes of VRE

vanA and vanB - carried on plasmids

vanA resistance level

high level of resistance

vanB resistance level

variable amount of resistance

Which Enterococcus species is most commonly isolated clinically?

Enterococcus faecalis

Which Enterococcus species is more likely to be vancomycin-resistant?

Enterococcus faecium

What is vanC-mediated resistance?

Intrinsic low-level vancomycin resistance encoded chromosomally, not plasmid-mediated

Which Enterococcus species naturally carry vanC?

Enterococcus gallinarum and Enterococcus casseliflavus

VRE testing methods - PCR

for vanA and vanB

VRE testing methods - vancomycin screening agar

6 ug of vacomycin in brain heart infusion agar with bile esculin

0.5 mcfarland and 10ul of suspension to plate

incubate 24h (if neg reincubate for 24h)

growth and bile esculin production shows resistance

MIC should be used in conjuction

what is high level aminoglycoside resistnace

a combination therapy of ampillin with aminoglycoside

Why is aminoglycoside used with ampicillin for enterococcal infections?

Synergy: Ampicillin weakens the cell wall → allows aminoglycoside (e.g., gentamicin) to enter → bactericidal effect

What alternative therapy is increasingly used instead of ampicillin + aminoglycoside?

Ampicillin + ceftriaxone → avoids HLAR problem

Which organisms commonly show inducible clindamycin resistance?

Staphylococcus spp., Streptococcus pneumoniae and beta-hemolytic
Streptococci

What lab profile suggests inducible clindamycin resistance?

Clindamycin susceptible (S) / Erythromycin resistant (R)

What are the two mechanisms behind inducible clindamycin resistance

msrA gene – efflux pump → clindamycin truly susceptible

erm gene – methylates ribosomal target → clindamycin can be induced to resistance

How do you differentiate msrA vs erm-mediated resistance?

D-test

Mechanism of inducible resistance in vitro?

Presence of erythromycin induces erm gene → ribosome altered → clindamycin resistance appears

D-test for inducible clindamycin resistance

0.5 mcfarland of organism and lawned

clindamycin and erythromycin place 15mm apart

incbate 16-18h

examined for “D” indicating the organism posses the erm gene and is inducible clyindamycin resistant

class A of beta lactamase producers

hydrolyze aminopenicillin and penicillinase-stable penicillins

inhibited by clavulanic acid

plasmid mediated and transfered between different genera

found in staphylococcus and enterococcus

include some ESBLS

class B of beta lactamase producers

need metal ion, zine, to hydrolyze beta-lactam ring

no inhibited by clavulanic acid

plasmid mediated

first found in S.maltophilia and transferred to P.aeruginosa, acinetobacter spp and comes enterbacteriacae

resistant to aminoglycosides and fluoroquinlones

class C of beta lactamase producers

cephalosporinase AmpC

crhomosomal mediated

require prior exposure to beta lactam

not inhibited by clavulanic acid

resistant to imipenem

found in most enterobacteriaseae

class D of beta lactamase producers

lease common beta - lactamase mech

not inhibited by clavulanic acid

active against carbapenems, cephalosportins, penicillins, and oxacillin’

found in P.aeruginosa, acinetobaccter spp. and few enterobacteriaceae

Ambler Classification of Beta-lactamases - Class A

serine

inhibited by clavulanic acid, tazobactam

Ambler Classification of Beta-lactamases -class B

metallo-beta-lactamase

requires metal ion (zinc) to hydrolyze beta-lactam ring

inhibit by chelator ex. EDTA and new beta lactamase inhibitors

Ambler Classification of Beta-lactamases - class C

serine

cephalosporinase AmpC

Ambler Classification of Beta-lactamases - Class D

weakly hydrolyze carbapenems

what genes are in ESBLs

TEM, SHV-1, CTX-M

ESBLs hydrolyze all what

penicillins, cephalosporins, monobactams

how do you treat ESBLs

cephamycin (cefoxitin, cefotetan) carbapenems

ESBLs are mediated by what

plasmid mediated and can be transferred to other organisms

Which four antibiotic disks are used in ESBL confirmation testing?

• Ceftazidime

• Ceftazidime + clavulanic acid

• Cefotaxime

• Cefotaxime + clavulanic acid

Why is clavulanic acid used in ESBL testing?

It inhibits ESBL enzymes, restoring antibiotic activity

How do you interpret a positive ESBL confirmation test?

≥5 mm increase in zone diameter with clavulanic acid compared to the drug alone

What does an increase in zone size with clavulanic acid indicate?

Presence of ESBL enzyme

what is AmpC

cephalosporinase producer

Which antibiotics does AmpC act on?

1st, 2nd, 3rd generation cephalosporins

Penicillins

Aztreonam