persistent and chronic infections

Question 1

if antibiotics were 100% effective then all infections would

Answer 1

cleared

Question 2

bacterial populations contain

Answer 2

peristers

Question 3

persister cells

Answer 3

phenotypic variants that constitute approx 1% of cells in stationary phase and biofilm culture. They are multi drug tolerant and largely responsible for the inability of antibiotics to completely eradicate infections

Question 4

when were persister cells first recognised

Answer 4

1940

Question 5

presence of persists cells

Answer 5

may be important in the aetiology of many recalcitrant infectious diseases

Question 6

when antibiotic are given

Answer 6

regular cells will decreases, until just persisters and resistant matts are left resistant mutants will continue to reproduce, whilst persister levels will never change

Question 7

when antibiotics are removed

Answer 7

persister cells are able to regrow

Question 8

why aren’t persistrcells resistant mutants

Answer 8

they won’t grow in the presence of antibiotics

Question 9

persister cells are not

Answer 9

genetically different

Question 10

formation of persister rapidly increases during

Answer 10

mid-exponential phase m several species, but the mechanism that underlies this remains a puzzle

Question 11

since persister cells are genetically identical to susceptible bacteria

Answer 11

they are phenotypic variants of the wild type

Question 12

how are persister cell isolated

Answer 12

by applying a lethal dose of antibiotics to a growing cult,.

Question 13

after removal of antibiotics persisters can regrow and give rise to

Answer 13

antibiotic sensitive cells that are genetically identical to the original population

Question 14

what indicates that the optimal individual strategy is not to enter into persistence

Answer 14

that fact that most E.coli cells in the stationary phase population are non persisters

Question 15

why is presser state thought to be altruistic behaviour

Answer 15

since being persister is not optimal and benefits kind

Question 16

persister cells in biofilms are able to

Answer 16

regrow after cessation of antibiotic therapy, whereas planktonic cells are not

Question 17

an approach to treat persisters

Answer 17

to serially reduce the conc of antibiotics thereby generating fewer numbers of persisters with each round of treatment

Question 18

how do we identify persisters from a population if they are non growing

Answer 18

• add antibiotics
• kill all non-persister cells
• will be left with just persister cell

Time consuming and microscopy is expensive

Question 19

FACS

Answer 19

fluorescence activating cell sorting

Question 20

FACS more

Answer 20

-seperates cells on size and colour- can show which populations show most fluorescence- the less fluorescent the more likely to be persistent.

Question 21

the less fluorescent the

Answer 21

more likely to be persistent

Question 22

how does FACS work

Answer 22

promotors will drive the transcription of GFP (edit promotes to make it active). Persister cels won’t produce GFP since they aren’t growing, therefore they can be seen in phase contrast (smaller), but will not fluoresce since they parent growing.

Question 23

fluorescence dilution

Answer 23

FD can be used to track non-replicating persister in infected hosts. Bacterial cells are loaded with fluorescent protein, once 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 mark for the absence of replication

Question 24

Why is the lack of FD a marker for the absence of replication

Answer 24

if persister, it won’t be replicating, so fluoresce should stay the same conc. If normal, then the fluorescence should fade after every division, until no fluoresce

Question 25

single cell analysis such as microscopy, flow cell cytometry or microfluidics can reveal

Answer 25

a diff picture with respect to the population

Question 26

it is thought that persister are produced by a

Answer 26

stochastic (random) procesws

Question 27

2 processes control persister formation

Answer 27

• a stochastic fluctuation in the lede, of persister proteins - a controlled, regulated mean level of expression of these proteins- dependent on the density of the pop. and other factors

Question 28

environment sets the baseline over

Answer 28

which stochastic fluctuation of expression will take place

Question 29

the simplest route to form a dormant persister cell might be through..

Answer 29

the over production of proteins that are toxic to the cell and inhibit growth-toxins

Question 30

Toxin antitoxin systems

Answer 30

plasmid or chromosomal located gene locus, consisting of toxin and antitoxin. Three classes of TA systems based on gene products

Question 31

type 2 TA system

Answer 31

gene products are proteins.

Free toxin binds to intracellular target to cause phenotypic change

Question 32

Antitoxin bind to form TA complex and prevents

Answer 32

toxin activity

Question 33

environment may degrade the anti toxin

Answer 33

the toxin is liberated and this will cause growth arrest- cell death

Question 34

toxins inhibit

Answer 34

DNA replication, protein or peptidoglycan stones to cause dormancy or cell death –> leads to growth arrest

Question 35

antitoxins are more ….. than toxins

Answer 35

unstable

Question 36

example of genes that caused growth arrest in E.coli

Answer 36

HicA

Question 37

HicA expression increased

Answer 37

persister frequencies

Question 38

reintroduction of HicB leads to

Answer 38

restoration of growth

Question 39

are all persisters dormant

Answer 39

evidence for various active mechanisms of antibiotic tolerance such as detoxification through efflux pumps or lower antibiotic take up

Question 40

persistance

Answer 40

the ability of bacteria to remain viable in the host for a prolonged period of time. Not to be confused with bacterial persistance

Question 41

bacterial persisters

Answer 41

cells that represent a subset of antibiotic tolerance; persisters are sub population of slow-growing or growth-arrested bacterial cells that ave a decreased susceptibility to killing by bactericidal antibiotics within an otherwise susceptible clonal population, owing to a low target activity or low antibiotic uptake that is induced by stress

Question 42

are all cells equal- which method should we consider

Answer 42

western blot of different BCG extracts

Question 43

BCG

Answer 43

Bacillus Calmette Guerin

Question 44

are all cells equal experiment

Answer 44

western bootof off diff BCG extracts probed with antibody against the surface protein MPB83

• on first appearance it seems that BCG Japan produces more MPB83 then BCG russia
• white, low producers
• grey, medium producers
• black, high producers

Question 45

microscopy, flow cell cytometry or microfluidics can

Answer 45

reveals a diff pict with respect to the population.

Question 46

single cell analysis techniques

Answer 46

microscopy, flow cell cytometry or microfluidics

Question 47

how are TA systems related to persister cell formation

Answer 47

Recent studies have demonstrated that gene loci known as toxin-antitoxin (TA) modules play a central role in the persister state. Under normal growth conditions, antitoxins potently inhibit the activities of the toxins. In contrast, under conditions of stress, the antitoxins are selectively degraded, freeing the toxins to inhibit essential cellular processes, such as DNA replication and protein translation. This inhibition results in rapid growth arrest.

Question 48

toxins that usually cause cell arrest but be

Answer 48

counteracted by an antitoxin- this occurs all the time

Question 49

antibiotics cause

Answer 49

antitoxins to be released from toxins, meaning they can have their potent effect own the cell, causing inhibition of DNA replication and protein translation- growth arrest