1) Persister bacterial cells are dormant, non-dividing cells that are tolerant to antibiotics but not resistant. They are genetically identical to actively growing cells. 2) Persister cells are found in all bacterial species and are enriched in biofilms, making chronic infections difficult to treat. They are responsible for treatment failure and relapse. 3) Persister cells form through spontaneous switching or in response to stress like nutrient starvation. Overexpressed toxin/antitoxin systems enable drug tolerance in persisters.

1. Persister bacterial
cells
Dr. Giuliani Francesco Ph.D
Molteni Therapeutics
BU riceca PDT
Mail: f.giuliani@moltenitherapeutics.it
Phone: +39 055 7361 - 239
Skype: Moltenitherap
Molteni Therapeutics S.R.L.
Via I. Barontini, 8 – Loc. Granatieri
50018 Scandicci (FI) Tel. +39 055 7361285
Website: www.moltenitherapeutics.it
Mail: info@moltenitherapeutics.it

2.  A persister cell has a global slowdown of metabolic
processes and does not divide
 Non resistant to antibiotic but simply TOLERANT
 Temporary phenotypic variants genetically identical
to major population
 They are still susceptible to the antibiotics
 Common to all bacterial species
 Common in Biofilm formation
 Responsible for the recalcitrance of chronic
infections
Persister bacterial cells
K. Lewis. (2010): Persister Cells. Annu. Rev. Microbiol. 64: 357-372

3. Few of these cells are formed in early
exponential phase, followed by a
sharp increase in persister cells
formation in mid-exponential phase,
reaching a maximum of
approximately 1% of cells forming
persisters in the non growing
stationary phase (frequency usually
10-4 – 10-6).
persisters formation: mainly originate from
stationary phase
Bacterial “lifecycle” in Lab

4. Importantly
Persisters are not formed in response to
antibiotic treatment
Keren, I., et al. (2004). Persister cells and tolerance to antimicrobials. FEMS Microbiol.
Lett. 230, 13–18,.

5. Mechanism
There are more than one mechanism to make persisters,
completely independent one another
 Spontaneous Persistence
Phenotypic switching of a few organisms to a dormant or protected
state occurs spontaneously and continuously in any growing
microbial population regardless of the presence of a drug.
 Environmentally induced
A number of environmental signals can modulate the level of
persistence in an isogenic microbial population.
 Heat shock
 Oxidative stress
 Nutrient starvation
 Population density
 DNA damage
 Quorum sensing

6. Stress-Response enable drug
persistence
There is increasing evidence for the active involvement
of various and interconnected intracellular stress
responses.
Most stress responses lead directly or indirectly to a slowing or
stalling of bacterial growth and division thus decreasing the
efficacy of the antibiotic treatment
K. Lewis and co-authors discovered, by transcriptome analysis,
that in persisters there are overexpressed genes that are present in
all chromosomes of bacteria and called toxin/antitoxin
Poole, K. (2012). Stress responses as determinants of antimicrobial resistance in
Gram-negative bacteria. Trends Microbiol. 20, 227–234.
K. Lewis. (2010): Persister Cells. Annu. Rev. Microbiol. 64: 357-372

7. Toxin/Antitoxin (TA) System
Toxin-antitoxin (TA) systems
typically consist of two genes
in an operon which encode a
stable toxin that disrupts an
essential cellular process
(e.g., translation via mRNA
degradation) and a labile
antitoxin (either RNA or a
protein) that prevents
toxicity.
X. Wang and T.K. Wood (2011): Toxin-Antitoxin System Influence biofilm and
Persister Cell Formation and the General Stress Response. Appl . Env. Microbiol.
77: 5577-832

8. By Joseph BIGGER, an army physician
“Penicillin did not have the ability to
sterilized an infection”
Discovery in 1944
JW Bigger (1944): Treatment of Staphylococcal infection with penicillin. The
Lancet Volume 244, 497-500

9. Bacterial resistance
 Including whole population
 Genetically determined

10. A state in which a sub-population of
DORMANT bacteria, or “PERSISTER”,
TOLERATE antibiotic treatment.
Chronic infections are often caused by pathogens
that are susceptible to antibiotics, but the disease
may be difficult or even impossible to eradicate with
antimicrobial therapy. Here is the paradox.
Bacterial tolerance

11. Resistance vs Tolerance
Tolerance works in a different way, not by
preventing antibiotic binding but by
interfering with the lethal action of
antimicrobial agents.
Keren, I., et al. (2004). Specialized persister cells and the mechanism of antibiotic
tolerance in Escherichia coli. J. Bacteriol. 186: 8172–8180

12. Resistance vs Tolerance
a | The antibiotic (pink) binds
to the target (blue) altering its
function, which causes cell
death.
b | The target of the antibiotic
has been altered so that it fails
to bind the antibiotic and the
cell becomes resistant to
treatment with the drug.
c | A different molecule (yellow)
inhibits the antibiotic target.
This prevents the antibiotic
from corrupting its functions,
resulting in tolerance.
K. Lewis. (2007): Persister Cells, dormancy and infectious disease. Nature. 5: 48-
56

13. K. Lewis. (2007): Persister Cells, dormancy and infectious disease. Nature. 5: 48-
56
Resistance vs Tolerance

14. M. Fauvart et al. (2011): Role of persister cells in chronic infections: Clinical
relevance and perspectives on anti-persister therapies. JMM. 60: 699-709
Resistance vs Tolerance

15. Schematic model of killing and
persistence kinetics during
antimicrobial therapy
Treatment of an initial population of
pathogens (I) causes killing of the
majority of cells (II) but fails to
eradicate a small subset of persisters
(III). When antibiotic pressure is
removed, persisters resume growth,
resulting in recurrent infection of
the host (IV). Retreatment results in
similar killing kinetics.
NR Cohen et al. (2013): Microbial Persistence and the Road to Drug Resistance. Cell
Host & Microbe. 13: 632-42
Resistance vs Tolerance

16. Biofilm and Persisters
 65% of infections are caused by Biofilms
 Can be formed by most, if not all, pathogens
 Infections highly recalcitrant to antibiotic treatment
However, planktonic cells that are derived from
biofilms are, in most cases, fully susceptible
 Importantly, biofilm do not actually grow in the
presence of elevated concentrations of antibiotics
Concentration of antibiotics required to kill biofilms are
not therapeutically achievable even if Biofilms do not
have increased resistance compared with planktonic cells

17. But how it is possible that a biofilm can
be resistant to killing by all
antimicrobials and not harbor a
resistance mechanism? Herein lies the
riddle of biofilm resistance.
The culprit-Persisters cells

18. Biofilm and Persisters
Persisters are highly enriched in biofilms.
The biofilm environment is advantageous to the
microbial population, however, when nutrients become
limited metabolic dormancy becomes the viable option.
Persisters are present at 0.1 – 1% in the biofilm of
Pseudomonas aeruginosa, Escherichia coli,
Staphylococcus aureus and Candida albicans.
Persisters may well represent the long-looked-for
explanation for biofilm tolerance to antibiotics.

19. How do biofilm avoid being killed?
K. Lewis. (2007): Persister Cells, dormancy and infectious disease. Nature. 5: 48-
56
Persisters are responsible for
multidrug tolerance of Biofilms
Initial treatment with antibiotic kills
normal cells (coloured green) in both
planktonic and biofilm populations. The
immune system kills planktonic
persisters (coloured pink), but the biofilm
persister cells (coloured pink) are
protected from the host defences by the
exopolymer matrix. After the antibiotic
concentration is reduced, persisters
resuscitate and repopulate the biofilm and
the infection relapses.

20. Implications
Persistence-enabling stress responses accelerate
acquisition of resistance
Persistence and cellular growth may not always be
incompatible (wakamoto et al 2013).
Persisters may represent a pool of adaptively evolving
organisms from which resistant mutants can emerge.
 Stress responses promote adaptive mutation
 Stress responses promote horizontal gene transfer
Y. Wakamoto et al. (2013). Dynamic persistence of antibiotic-stressed mycobacteria.
Science 339, 91–95.

21. Drug Persistence in Human Disease
Persistence is widespread and has been described in
bacteria and fungi
Persistence contributes to the pathogenesis of several human
infections that required protracted treatment and that relapsed
after therapy
 Cystis Fibrosis pneumonia (CF)
 Candidiasis
 Tubercolosis

22. Drug Persistence in Human Disease
NR Cohen et al. (2013): Microbial Persistence and the Road to Drug Resistance. Cell
Host & Microbe. 13: 632-42
CF, Cystic Fibrosis.
a The range of treatment duration,
b The typical numbers of drugs required to achieve apparent eradication of the organism.

23. Persisters and Antimicrobial Therapy
“ the more we understand about
persisters and the mechanisms of
their formation, the less
understanding of those mechanisms
is useful in designing drugs “

24. It is possible to develop a single-compound
antipersister antibiotic?
The perfect antibiotic
The pro-antibiotic is benign, but a
bacterial enzyme converts it into a
reactive antibiotic in the cytoplasm.
The active molecule does not leave
the cytoplasm (owing to increased
polarity), and attaches covalently to
many targets, thereby killing the
cell. Irreversible binding to the
targets prevents the antibiotic from
multidrug resistance efflux.
K. Lewis. (2007): Persister Cells, dormancy and infectious disease. Nature. 5: 48-
56

25. Photodynamic therapy may act as the
“perfect antibiotic”
 PS molecules active only if illuminated
 Broad spectrum
 Multitarget
 Does not induce mechanism of resistance*
hard to prove sperimentally
 Unculturable in vitro
 Their temporary phenotype precludes introducing
them into an animal model of infection
*F Giuliani et al. (2010): In Vitro Resistance Selection Studies of RLP068/Cl, a New
Zn(II) Phthalocyanine Suitable for Antimicrobial Photodynamic Therapy. AAC. 54:
637-42

26. However..........
Tegos and co-workers demonstrate that phenothiazinium-
based PSs (MB, TBO & DMMB) are substrate of multidrug
–resistance efflux pumps in bacteria.
This is not the case for the other PSs
including phthalocyanines.
GP Tegos et al. (2006): Phenothiazinium antimicrobial photosensitizers are substrates
of bacterial multidrug resistant pumps. AAC. 50: 196-203
GP Tegos et al. (2008): Inhibitors of bacterial multidrug efflux pumps potentiate
antimicrobial photoinactivation. AAC. 52: 3202-3209

27. 1
2 3
4
5
6
7
8
8
Experiment proposal
1. Bacterial inoculum
2. Grow the culture16-18 hours at 37°C
under aerobic conditions
3. Leave it 3-4 hours in the presence of a
proper antimicrobial agent (5 × MIC)
4. Add 1.5 ml clarified medium into an
eppendorf tube (Persisters?)
5. Eventually persisters cells harvested by
centrifugation, washed twice with FS and
resuspended in 1.0 ml of the same buffer
6. Incubation in the dark with a 2 × MBC
RLP068 concentration
7. Illumination
8. Serial dilution and plating on solid
growth medium.

28. Expected result
 Bacterial growth in control plates as
from literature
 Complete sterilization of the plates
coming from the PDT treated sample

29. Questions?

30. Empty your mind.
Be formless, shapeless
like water.
You put water in a cup
It becomes the cup.
You put water in a bottle
It becomes the bottle.
You put water in a teapot
It becomes the teapot.
Water can flow
Or it can crash.
Be water my friend