Mechanisms of Antibiotic Resistance (gram negative bacteria)

What is high-level resistance (3)?

• target-specific mutations / transferable elements

• specific to a single antibiotic class

• clinical failure

What is low-level resistance (4)?

• upregulation of intrinsic loci

• permeability modulation

• reduced susceptibility to multiple classes

• microbial survival under host and drug pressure

What are some possible mechanisms bacteria can utilise to increase resistance (5)?

• overexpress membrane pumps (pump out antibiotics)

• develop antibiotic-modifying enzymes

• mutate genes in antibiotic targets (cannot bind)

• modify LPS

• downregulate membrane pumps (decrease antibiotic entry)

How can bacteria develop resistance to beta-lactams class antibiotics (3)?

• produce beta-lactamases

• perturbation of penicillin binding proteins

• change cell permeability (membrane pump expression)

What are beta-lactamases and how do they work (3)?

• enzymes that degrade beta-lactams antibiotics

• located in periplasmic space

• usually plasmid mediated

Which bacteria have shown chromosomal beta-lactamase expression?

Enterobacter spp.

How does chromosomal beta-lactamase expression (and so resistance) work in the case of repressor mutation (3)?

• beta-lactamase gene has a promoter and repressor

• normally, repressor blocks action of RNA polymerase

• mutations in repressor allow transcription = resistance

How can antibiotic presence lead to chromosomal beta-lactamase expression (and so antibiotic resistance) (2)?

• antibiotic can limit the action of the repressor (can remove it)

• repressor not present on beta-lactamase gene = expression and resistance

What are the 2 possible mechanisms of chromosomal beta-lactamase expression?

• induction (interference with repressor)

• de-repression (repressor mutated)

Where are resistance genes found (naturally / not acquired) (2)?

• organism that produces an antibiotic must also be resistant to it

• resistance genes upstream of antibiotic gene cluster

How do resistance genes move from the chromosome into a plasmid?

gene capture mechanisms excise the gene

What species has resistance gene excision from the chromosome into a plasmid been observed in?

Streptomyces spp.

How do beta-lactamases act on beta-lactams antibiotics to produce resistance (2)?

• cleave central ring and flips the molecule

• conformational change = can no longer inhibit bacteria

What is vertical resistance spread?

cross-infection from patient to patient (failure of containment)

What is horizontal resistance spread?

formulary - external issue from antibiotic pressure to spread the resistance plasmid

What are the 4 main classes of beta-lactams antibiotics?

• penicillins

• cephalosporins (1st - 4th & 5th generations)

• carbapenems

• monobactams

How are the different beta-lactams drugs characterised (4)?

• class and active site (e.g. A-serine)

• substrate (e.g. penicillins, carbapenems etc)

• chromosomal (gram positive, negative or both)

• plasmid (gram positive, negative or both)

How do beta-lactamases act on amoxycillin (2)?

• targets amide ring

• flips side chain = cannot bind targets

What beta-lactamase targets amoxycillin?

TEM-1 beta-lactamase

What is an example of a large molecule cephalosporin?

ceftazidime

What are 2 examples of small molecule cephalosporins?

• ceftriaxone

• cefotaxime

Why do small molecule cephalosporins have a slower rate of resistance emergence compared to large molecules (2)?

• small = quicker permeation

• can surmount challenge of beta-lactamases in periplasmic space

What is an example of a cephalosporin resistant bacteria that is becoming increasingly common in Europe?

Klebsiella pneumoniae

How do you treat a bacteria that is resistant to cephalosporins (2)?

• use the next generation up of beta lactams (e.g. 3rd gen cephalosporin if resistant to 2nd gen)

• OR use a non beta-lactams class antibiotic

Why has there been an observed concurrent increase to both 3rd gen cephalosporins and fluoroquinolones in E.coli from blood and CSF?

clonal spread of one type of microbe (ST131 / strain ‘A‘)

Which E.coli strain is resistant to 3rd generation cephalosporins and fluoroquinolones and how (3)?

• ST131 / strain ‘A‘

• contains CTX-M-15 enzyme (3rd generation cephalosporin resistance)

• chromosome mutation in topoisomerase (fluoroquniolone resistance)

What are your options for a bacteria resistant to all cephalosporin classes?

the next class up - carbapenems (the big guns)

How can one plasmid acquire multiple resistance genes (process) (4)?

1. CTX-M gene with an insertion sequence

2. co-opted into an integron (with other genes present)

3. co-opted into a transposon (even more other genes)

4. moved into a plasmid or chromosome

What is the name of the process by which plasmids acquire multiple resistance genes?

DNA capture

Why is resistance on plasmids so concerning (3)?

• amplification of genes in plasmid (each has multiple different resistance genes, not just one)

• plasmid copy number

• rapid movement of plasmids

What is our last line of antibiotics?

carbapenems

How did E.coli ST131 (Strain ‘A’) begin to develop carbapenem resistance (2)?

• loss of a porin (antibiotic cannot enter)

• NOT acquisition of a new gene (as may be expected)

Which antibiotic remains effective against most carbapenem resistant drugs?

colistin (active vs >90% MDR isolates)

What is the name of the beta lactamase gene discovered in 2009 in India that can break down all known beta-lactams?

NDM-1 (can break down carbapenems)

What is a good example of the global spread of resistance (3)?

• NDM-1 (can break down every beta-lactams)

• discovered in India 2009 and now globally distributed

• ubiquitous (found in many different bacteria)

What is the structure of a transposon (a ‘jumping gene’)?

gene to be moved flanked by 2 insertion sequences

How do plasmids acquire multiple resistance genes (process name)?

transposition (‘jumping genes‘)

What are the 2 methods of gene transposition?

• conservative transposition

• replicative transposition

How does transposition work (generally) and why is this such as challenge (3)?

• plasmid taken up and donates resistance genes to chromosome

• chromosome donates genes to new plasmid

• difficult to track and target

How does conservative transposition work (4)?

• transposon is excised from one site in DNA and inserted into a new site

• NO transposon duplication occurs

• donor DNA loses the transposon

• mediated by transposase enzyme

How does replicative transposition work (4)?

• transposon is replicated - copy is inserted into the target DNA and original remains in donor

• forms a temporary co-integrate structure - donor and target DNA are joined

• requires transposase and resolvase enzymes to separate the co-integrate

• results in 2 copies of transposon

What is the result of conservative transposition (2)?

• one transposon in the target replicon

• no copy in the donor replicon

What is the result of replicative transposition (2)?

• two copies of transposon

• one in target and one in donor

What is ‘natural’ genetic engineering (2)?

• transposition via transposons and integrons

• i.e. gene capture and expression