34 Attacking the enemy: antibiotic resistance

Question 1

What are general ways in which mechanisms of resistance develop?

Answer 1

Drug target modified so can no longer bind

Reduced cell wall/ membrane permeability

Efflux drug

Enzyme destruction of drug

Alternate metabolic pathway

Question 2

Resistance mechanism -

Drug target modified so can no longer bind

Some mutations can involve single nucleotide changes, so can arise rapidly. But may not spread as quickly as they are chromosomal in nature (not plasmid)

What drug targets can be modified?

Answer 2

Cell surface protein

Ribosome

Topoisomerase enzymes

Question 3

How does MRSA change its cell surface protein to become resistant?

Answer 3

gene from mec family encodes for different penicillin-binding-protein 2a

Question 4

Hows does glycopeptide resistance arise?

Answer 4

Mutation in peptidoglycan precursor structure causes subsitution with d-alanyl-d-alanine termination

So glyocpeptides cannot bind

VanA gene confers high level resistance vanc/ teicoplanin

Van B gene confers low level resistance to vancomycin only

Question 5

Ribosomal mutation can prevent drug binding.

What are common mutations?

Answer 5

Target modification by methylation of 23S RNA in the 50S subunit

erm gene codes for this

Encodes resistance to MLSb - macrolides, lincosamides, streptogrammin B and sometimes linezolid

Question 6

What mutation confers resistance in topoisomerase?

Answer 6

GyrA gene mutation renders fluoroquinolones ineffective

Question 7

Reduction in cell wall/ membrane permeability is relatively uncommon cause of resistance.

What is an example of this?

Answer 7

Pseudomonas - porin loss can cause beta-lactam resistance

Question 8

What are examples of genes and species which increase drug efflux?

Answer 8

mefA gene streptococcus - efflux macrolides

tetk gene streptococcus - efflux tetracyclines

Mex gene pseudomonas - efflux beta-lactams, fluoroquinolones, aminoglycosides

Acr gene E. coli - effluoc beta-lactams, fluoroquinolones, trimethoprim

Question 9

Metabolic pathways can be altered to provide resistance

How do trimethoprim/ sulphonamide drugs become inactviated?

Answer 9

New folate synthesis pathway enables bypass

Question 10

Metabolic pathways can be altered to provide resistance

How does metronidazole resistance occur?

Answer 10

Inactive until chemically reduced

Reduced form cytotoxic, damaging DNA

nim family of genes, particularly in bacteroides species, encodes alternative enzyme which can process metronidazole into less cytotoxic form

Question 11

Metabolic pathways can be altered to provide resistance

How does nitrofurantoin resistance occur?

Answer 11

Nitrofuranotin is inactive until chemically reduced

Reduced form cytotoxic, damaging DNA

E. coli mutates so has alternative metabolic pathway, producing inert drug

Question 12

Patient has colectomy, and post-op infection. Enterococcus grown, resistant, and has erm gene.

which antibacterial will retain its activity?

clarithromycin
clindamicin
erythrmycin
pristinamycin
vancomycin

Answer 12

vancomycin

erm genes codes for MLSb resistance

Question 13

What is the Ambler classification used for?

Answer 13

Molecular method used to classify beta-lactamases

Classes
A-D

Question 14

What are classes of Ambler Classification

A-D

Answer 14

A -
Narrow spectrum beta-lactamases - penicillin
Extended spectrum beta-lactamases - ESBL
Serine carbapenemases - hydrolyse carbapenems

B -
mettalo-beta-lactamases - hydrolyse carbapenemas

C-
cephalosporinases - hydrolyse cephamycins (AmpC)

D -
OXA-type enzymes

Question 15

What is function of AmpC enzymes?

What antibiotics are inhibited?

Answer 15

Ambler class C

Hydrolyse cephalosporins and penicillins.

Not inhibited by beta-lactamase inhibitor clavulanic acid.

Resistant -
co-amoxiclav
tazocin
carbapenems
ceftriaxone/ ceftaz/ cefotazime

Question 16

Patient initially improves on antibiotics, then deteriorates. AmpC gene detected

How is this explained?

Answer 16

If patient given co-amoxiclav/ tazocin for these infections, may initially improve.

Then AmpC genes switch on, and resistant strain begins to multiply. Patient then deteriorates

If cefoxitin resistant in vitro, that is indication that has AmpC gene. Even if sample sensitive to co-amoxiclav/ tazocin, do not use these are resistant strain will multiply

Question 17

what is function of ESBL enzyme?

Which antibiotics are inhibited?

Answer 17

Ambler class A

hydrolyse broader range of beta-lactams, including cephalosporins, monobactams

Tazocin
Cefotaxime - 4th generation
Aztreonam (memorise as group A Ambler class)

Often sensitive to co-amoxiclav, but better to use carbapenem as always sensitive

Question 18

Genetically speaking, where are ESBL and AmpC genes carried?

Answer 18

ESBL - plasmid - so can spread quickly

AmpC - chromosomal. Occasionally plasmid

Question 19

Following urine culture, and antibiotic susceptibility testing, when are isolates tested for -

ESBL

AmpC resistance

CPE/ CPO

Answer 19

ESBL - Any evidence of Cefpodoxime (3rd generation) resistance triggers further testing

AmpC - cefotaxime/ ceftazidime and Cefpodoxime resistance

CPE/ CPO -

• Eneterobacteriaceae - any evidence of ertapenem/ meropenem resistance
• Pseudomonas - meropenem/ ceftaz/ tazocin resistance

Question 20

Urine culture is resistant to cefpodoxime. You decide to check for ESBL production.

How is this performed?

Answer 20

Combination disk test - based on premise that most ESBLs are inhibited by clavulanic acid (AmpC are not inhibted by clavulanic acid)

Add disc with ceftazidime/ cefotaxime/ cefepime

Add disc with ceftazidime/ cefotaxime/ cefepime + clavulanic acid

Clavulanic acid inhibits beta-lactamases. If inhibited growth around this disc >5mm, and growth around other disc, suggests ESBL (as resistant to the ceftazidime/ cefotaxime)

Question 21

Which organisms have -

• plasmid encoded AmpC
• intrinsic chromosomal AmpC

Answer 21

- plasmid encoded AmpC
E. coli
Klebsiella
Proteus
Salmonella

- intrinsic chromosomal AmpC
Enterobacter
Citrobacter
Morganella
Providencia
Serratia

Question 22

Urine culture is resistant to cefpodoxime, and ceftazidime. You decide to check for AmpC production.

How is this performed?

Answer 22

Add cloxacillin disc

If inhibited by cloxacillin, suggests AmpC production

If not inhibited by cloxacillin, then resistance seen is via other mechanism

cannot use clavulanic acid test, as AmpC not inhibited by this