L20

Question 1

Five main bacterial antibiotic resistance mechanisms

Answer 1

1. Altered antibiotic target
2. Inactivate antibiotic
   a. Degrading the antibiotic
   b. Chemically modify antibiotic
3. Reduced antibiotic accumulation
4. Alternate pathway to bypass the inhibited one

Question 2

1. Altering the target of the antibiotic

Answer 2

• Target Protection - block access
• Target Modification - mod target
• Insensitive Functional Target - alt enzyme

Question 3

Target Protection

Answer 3

• Resistance protein blocks access of antibiotic target

Question 4

Target Modification

Answer 4

–Resistance enzyme modifies antibiotic target
–e.g. vancomycin R - arises when bacteria change the terminal D-ala in the pentapeptide of peptidoglycan into D-serine or D-Lactate, so vancomycin cannot bind

Question 5

Insensitive Functional Target

Answer 5

–Resistance protein is an alternative enzyme that is insensitive to antibiotic
–e.g. β-lactam R - MRSA produces an alternative PBP MecA which has low binding affinity for all β-lactams

Question 6

2a. Degrading the antibiotic

Answer 6

–e.g. hydrolysis of the β-lactam ring of penicillins and cephalosporins by penicillinase (β-lactamase) enzymes

Question 7

2b. Modifying the antibiotic

Answer 7

• Chemically altering antibiotic
• e.g. acetyltransferase adds an acetyl group to aminoglycosides or chloramphenicol, inactivating them

Question 8

3. Reduced antibiotic accumulation
   * Antibiotic efflux

Answer 8

–Rapid extrusion of antibiotic via efflux pump –e.g. multidrug exporter RND system of Gram negs can pump out quinolones, tetracycline and chloramphenicol
–extrusion is energy-dependent
–Note: bacteria can also reduce uptake

Question 9

4. Alternate pathway to bypass the inhibited one
   * Target bypass

Answer 9

–e.g. Enterococci are intrinsically resistant to sulphonamides because they are able to use pre-formed folic acid from their environment (unlike other bacteria)
–Group A Streptococcus reduced folate transporter

Question 10

• Vertical gene transfer

Answer 10

transfer of genes from parent to progeny
–bacteria have this, but as they don’t reproduce sexually so only limited genetic variation (via mutation)

Question 11

• Horizontal gene transfer (HGT)

Answer 11

transfer of genes from one independent, mature organism to another
–HGT is of major importance in bacterial evolution, including the gain of antibiotic-resistance genes

Question 12

Horizontal gene transfer is a one way process [t/f]

Answer 12

True

Question 13

How is HGT a one way process?

Answer 13

• Donor DNA enters a recipient cell
• If donor DNA contains genes already present in recipient, recipient
  becomes temporarily a partial diploid for those genes
• The donor DNA then has four possible fates in recipient cell
  –Integration via recombination with the recipient’s genome
  –Self-replication of the donor DNA (if plasmid)
  –No replication and loss of donor DNA
  –Degradation of donor DNA

Question 14

The donor DNA then has four possible fates in recipient cell if genes are present in recipient already

Answer 14

–Integration via recombination with the recipient’s genome
–Self-replication of the donor DNA (if plasmid)
–No replication and loss of donor DNA
–Degradation of donor DNA

Question 15

Mobile genetic elements:

Answer 15

DNA segments that can move to different locations on genomes and/or move from cell to cell. Includes
1. Plasmids
2. Transposable element
3. Integron

Question 16

Mobile genetic elements: Plasmid

Answer 16

double-stranded DNA molecule, usually circular, that can exist and replicate independently of the chromosome

Question 17

• R plasmid/ Resistance plasmid:

Answer 17

a plasmid bearing one or more antibiotic-resistance genes

Question 18

The first plasmid discovered was the

Answer 18

F plasmid
(or F factor)

Question 19

Mobile genetic elements) Transposable element

Answer 19

small linear DNA molecule that carries genes for transposition and thus can move around the genome, or from one genome to another (e.g. from plasmid to chromosome) via recombination

Question 20

Transposon

Answer 20

also carries other genes such as antibiotic resistance genes; is found as part of a bacterial chromosome or a plasmid, cannot self-replicate

Question 21

Mobile genetic elements: Integron

Answer 21

very small linear DNA molecule which can capture even smaller (~1kb) “gene cassettes” encoding antibiotic resistance; is found as part of a transposon or a plasmid, cannot self-replicate

Question 22

R plasmids encode antibiotic resistance;

Answer 22

• dis japan
• Some R have 1 res gene some have more
• Built up from smaller mobile genetic elements (MGEs)
• gene cassette encoding antibiotic resistance inserts into integron which inserts into transposon which in turn inserts into a plasmid
• R plasmids can spread throughout a bacterial population (by conjugation, transduction or transformation) and some can also spread between species

Question 23

gene cassette encoding antibiotic resistance

Answer 23

gene cassette encoding antibiotic resistance inserts into integron which inserts into transposon which in turn inserts into a plasmid

Question 24

R plasmids can spread throughout a bacterial population (by _____) and some can also spread between species

Answer 24

conjugation, transduction or transformation

Question 25

There are three main mechanisms of horizontal gene transfer (HGT) in bacteria

Answer 25

1. Conjugation 2. Transduction 3. Transformation *Using these mechanisms, antibiotic resistance genes can spread rapidly through bacterial populations

Question 26

Conjugation (HGT)

Answer 26

gene transfer mediated by plasmids that requires contact between cells

Question 27

Transduction (HGT)

Answer 27

gene transfer mediated by bacteriophages (viruses of bacteria)

Question 28

Transformation (HGT)

Answer 28

the uptake of a piece of free DNA by a cell

Question 29

What is an example of a conjugative plasmid

Answer 29

* F plasmids * Some R plasmids are conjugative plasmids, thus conjugation is a major route for the spread of antibiotic resistance genes among bacteria

Question 30

Conjugation with F plasmids

Answer 30

1. F plasmid encodes proteins to build sex pilus and protein systems (relaxosome, transferosome/Type 4 Secretion System) that perform DNA transfer 2. Sex pilus tip binds to recipient cell. Pilus retracts, pulling donor and recipient cells together to make contact and form a mating pore 3. Plasmid is nicked (one strand is cut), rolling circle replication of plasmid DNA starts. 4. Both cells recircularize plasmids, synth second strands to form double-stranded plasmids, and form pili; both cells now viable donors in conjugation.

Question 31

Transduction with bacteriophages (viruses of bacteria) includes two groups

Answer 31

1. Virulent phages: only have a lytic cycle 2. Temperate phages have a lytic cycle, or a lysogenic cycle

Question 32

Virulent phages:

Answer 32

only have a lytic cycle

Question 33

Temperate phages

Answer 33

have a lytic cycle, or a lysogenic cycle

Question 34

Virulent Phages lytic cycle:

Answer 34

* Phage injects DNA into cytoplasm * Phage DNA cause synth of new phages * Phage heads and tails made and copies of DNA genome packaged into them * cell lyse and release new phages that bind to bacterial cells

Question 35

Temperate Phages * Lysogenic cycle

Answer 35

where phage DNA integrates into bacterial host’s chromosome to form a **lysogen**. Phage DNA is inactive (prophage) and replicated along with chromosome; host cell does not lyse and grows normally.

Question 36

Phage DNA is inactive

Answer 36

prophage

Question 37

prophage induction occurs when

Answer 37

Under environmental stress

Question 38

once prophage induction happens:

Answer 38

prophage DNA is activated and excised from chromosome, replicates, and lytic cycle resumes – killing the cell

Question 39

Prophage induction:

Answer 39

exposure to stress such as UV light triggers activation of the prophage and its excision from the host chromosome – lytic cycle resumes

Question 40

Lysogenic cycle

Answer 40

* When phage inject DNA into cytoplasm * Phage DNA integrates into host chromosome * prophage DNA copied when cell divides * Prophage induction - activate prophage and excision from host to continue cycle

Question 41

Generalized transduction def

Answer 41

transfer of any genes from donor

Question 42

–Specialized transduction def

Answer 42

transfer of only genes adjacent to prophage

Question 43

Generalised transduction

Answer 43

* Occurs during the lytic cycle of virulent and temperate phages * Bacterial genes are packaged by mistake into a phage head (capsid). Phage carries only bacterial genes (i.e. no phage genes). * Any bacterial gene can be transduced, including antibiotic resistance genes

Question 44

Specialized transduction

Answer 44

* Occurs during the lysogenic cycle of *temperate* phages only * When a prophage is induced to excise from the host chromosome, excision is sometimes done incorrectly * resulting p. genome contains portions of the bacterial chromosome next to the prophage’s integration site in host chromosome * thus transducing phage *carries phage genes plus some bacterial genes*, which it will inject into its next host cell to form a transductant * *only certain bacterial genes* will be in the right location to be picked up & transduced by the temperate phage (i.e. tnext to the prophage)

Question 45

Specialized transduction is an important mechanism for ≈___ in bacterial evolution, as prophages can often carry virulence genes. Probably less significant in antibiotic resistance gene transfer.

Answer 45

Specialized transduction

Question 46

Transformation def

Answer 46

* Uptake of a piece of free DNA by a cell

Question 47

Transformation (HGT)

Answer 47

* Both chromosomal DNA and plasmid DNA transformed * Bacteria use complex DNA uptake systems to move DNA through b. cell envelopes * Natural transformation occurs when bacteria lyse and release DNA into environment - in soill, aquatic environments, in vivo during infections. Biofilms - Important for HGT

Question 48

* Natural transformation best studied in Streptococcus, Bacillus and Neisseria –artificial transformation carried out in molecular biology labs to induce other bacteria like ____ to take up DNA

Answer 48

E. coli

Question 49

____ (model conjugative plasmid), _____are built up from smaller Mobile Genetic Elements (MGEs): gene cassettes, integrons, transposons, and may be conjugative

Answer 49

F plasmid R plasmids