Persistent Infections Flashcards
If antibiotics and the immune system were a 100% effective
All infections would be cleared
What is a persister?
A phenotypic variant that constitutes about 1% of cells in stationary phase and biofilms
What is a persister cell?
Dormant variants of regular cells that form stochastically (randomly) in microbial populations and are highly tolerant to antibiotics
The presence of persister cells
Might be important in the aetiology of many recalcitrant infectious diseases
The formation of persisters rapidly increases during
Mid- exponential phase in several species, but the mechanism that underlies this remains unknown
Bacterial growth
Log phase, stationary phase, death phase
Unlike resistant cells, persisters are
genetically identical to susceptible bacteria, constituting phenotypic variants of the wild type
Persister cells are isolated by
Applying a lethal dose of antibiotics to a growing culture
A biphasic killing curve is
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
After removal of antibiotic persisters can
regrow and give rise to antibiotic-sensitive cells that are genetically identical to the original population
The persister state is
An altruistic behaviour benefiting the kin
Most cells do not become persisters
Averaged data sets
Do not show single cell variation within a population
Single cell analysis can be done by
Microscopy, flow cell cytometry or microfluidics
Persister cells are formed by
A stochastic process
Two processes control persister formation
- Stochastic fluctuation in the level of persister proteins
2. Controlled, regulated mean level of expression of these proteins
Regulation of persister proteins
Is dependent on the density of the population, and probably on several other factors as well
What ultimately controls stochastic persister fluctuation
The environment
How do we identify persisters?
- Treat a culture with ABs (cells left alive are persisters)
- Microfluidics
- FACS (fluorescent activated cell sorting)
- FD (fluorescence dilution)
FACS
Fluorescent activated cell sorting
A form of flow cytometry using degradable GFP
FD
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
Microfluidics
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
Flow cytometry
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
Persister cells are formed by
An overproduction of proteins that are toxic to the cell and inhibit growth - toxins
Persister toxin expression is
under regulatory control
Persister cell toxins are an example of
TA (Toxin Anti toxin) systems
TA systems are
- On a Plasmid or chromosomal gene locus
- Locus consists of toxin and antitoxin gene
- Free toxin binds to intracellular target that causes phenotypic change
- Antitoxin binds toxin to form TA complex and prevents toxin activity
How many TA classes are there
Three classes of TA systems based on gene products
Type 2 systems most studied
Products are proteins
What do toxins do
- Inhibit DNA replication
- Inhibit protein/peptidoglycan synthesis
- Others are mRNAases
The antitoxin is
More unstable
TA systems are
Stress induced
In E.coli, HicA expression leads to
Growth arrest (toxin)
In E.coli, HicB expression leads to
Restoration of growth (antitoxin)
HicA expression increases
Persister frequencies
Generation of persisters is also dependent upon
Cell density
RelE mediated dormancy in E.coli is enhanced at
High cell density
RelE expression is controlled by
An inducible rhamnose promoter
Quorum Sensing is
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
2’ Amino-acetophenone is a
Quorum sensing molecule produced by Pseudomonas aeruginosa
Why do antibiotics not affect persisters?
- They are not growing so targets ineffective
- They have pumps/efflux
- Lower AB uptake
- Other mechanisms
Persisters in biofilms are
Able to regrow after cessation of antibiotic therapy
Persisters are treated by
Serially reducing the concentration of antibiotics thereby generating fewer numbers of persisters with each round of treatment
Treating persisters is a problem because
The FDA only requires a new antibiotic to show efficacy against actively growing cells
