Persistent Infections

Question 1

If antibiotics and the immune system were a 100% effective

Answer 1

All infections would be cleared

Question 2

What is a persister?

Answer 2

A phenotypic variant that constitutes about 1% of cells in stationary phase and biofilms

Question 3

What is a persister cell?

Answer 3

Dormant variants of regular cells that form stochastically (randomly) in microbial populations and are highly tolerant to antibiotics

Question 4

The presence of persister cells

Answer 4

Might be important in the aetiology of many recalcitrant infectious diseases

Question 5

The formation of persisters rapidly increases during

Answer 5

Mid- exponential phase in several species, but the mechanism that underlies this remains unknown

Question 6

Bacterial growth

Answer 6

Log phase, stationary phase, death phase

Question 7

Unlike resistant cells, persisters are

Answer 7

genetically identical to susceptible bacteria, constituting phenotypic variants of the wild type

Question 8

Persister cells are isolated by

Answer 8

Applying a lethal dose of antibiotics to a growing culture

Question 9

A biphasic killing curve is

Answer 9

The 1st slope of the initial phase of killing represents the rapid death of the sensitive population
The 2nd slope of the second phase represents the much slower death of persisters

Question 10

After removal of antibiotic persisters can

Answer 10

regrow and give rise to antibiotic-sensitive cells that are genetically identical to the original population

Question 11

The persister state is

Answer 11

An altruistic behaviour benefiting the kin

Most cells do not become persisters

Question 12

Averaged data sets

Answer 12

Do not show single cell variation within a population

Question 13

Single cell analysis can be done by

Answer 13

Microscopy, flow cell cytometry or microfluidics

Question 14

Persister cells are formed by

Answer 14

A stochastic process

Question 15

Two processes control persister formation

Answer 15

1. Stochastic fluctuation in the level of persister proteins

2. Controlled, regulated mean level of expression of these proteins

Question 16

Regulation of persister proteins

Answer 16

Is dependent on the density of the population, and probably on several other factors as well

Question 17

What ultimately controls stochastic persister fluctuation

Answer 17

The environment

Question 18

How do we identify persisters?

Answer 18

1. Treat a culture with ABs (cells left alive are persisters)
2. Microfluidics
3. FACS (fluorescent activated cell sorting)
4. FD (fluorescence dilution)

Question 19

FACS

Answer 19

Fluorescent activated cell sorting

A form of flow cytometry using degradable GFP

Question 20

FD

Answer 20

Fluorescence dilution
Bacterial cells are loaded with a fluorescent protein
After the induction is switched off dilution of the pre- formed pool of fluorescent protein will report the extent of bacterial replication
The lack of FD is a marker for the absence of replication

Question 21

Microfluidics

Answer 21

The science of manipulating and controlling fluids, usually in the range of microliters (10-6) to picoliters (10-12), in networks of channels with dimensions from tens to hundreds of micrometers

Question 22

Flow cytometry

Answer 22

A technique used to detect and measure physical and chemical characteristics of a population of cells or particles.
A sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument. The sample is focused to ideally flow one cell at a time through a laser beam and the light scattered is characteristic to the cells and their components. Cells are often labeled with fluorescent markers so that light is first absorbed and then emitted in a band of wavelengths

Question 23

Persister cells are formed by

Answer 23

An overproduction of proteins that are toxic to the cell and inhibit growth - toxins

Question 24

Persister toxin expression is

Answer 24

under regulatory control

Question 25

Persister cell toxins are an example of

Answer 25

TA (Toxin Anti toxin) systems

Question 26

TA systems are

Answer 26

1. On a Plasmid or chromosomal gene locus
2. Locus consists of toxin and antitoxin gene
3. Free toxin binds to intracellular target that causes phenotypic change
4. Antitoxin binds toxin to form TA complex and prevents toxin activity

Question 27

How many TA classes are there

Answer 27

Three classes of TA systems based on gene products
Type 2 systems most studied
Products are proteins

Question 28

What do toxins do

Answer 28

1. Inhibit DNA replication
2. Inhibit protein/peptidoglycan synthesis
3. Others are mRNAases

Question 29

The antitoxin is

Answer 29

More unstable

Question 30

TA systems are

Answer 30

Stress induced

Question 31

In E.coli, HicA expression leads to

Answer 31

Growth arrest (toxin)

Question 32

In E.coli, HicB expression leads to

Answer 32

Restoration of growth (antitoxin)

Question 33

HicA expression increases

Answer 33

Persister frequencies

Question 34

Generation of persisters is also dependent upon

Answer 34

Cell density

Question 35

RelE mediated dormancy in E.coli is enhanced at

Answer 35

High cell density

Question 36

RelE expression is controlled by

Answer 36

An inducible rhamnose promoter

Question 37

Quorum Sensing is

Answer 37

The regulation of gene expression in response to fluctuations in cell-population density

Quorum sensing bacteria produce and release chemical signal molecules called autoinducers that increase in concentration as a function of cell density

Question 38

2’ Amino-acetophenone is a

Answer 38

Quorum sensing molecule produced by Pseudomonas aeruginosa

Question 39

Why do antibiotics not affect persisters?

Answer 39

1. They are not growing so targets ineffective
2. They have pumps/efflux
3. Lower AB uptake
4. Other mechanisms

Question 40

Persisters in biofilms are

Answer 40

Able to regrow after cessation of antibiotic therapy

Question 41

Persisters are treated by

Answer 41

Serially reducing the concentration of antibiotics thereby generating fewer numbers of persisters with each round of treatment

Question 42

Treating persisters is a problem because

Answer 42

The FDA only requires a new antibiotic to show efficacy against actively growing cells