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Persistercells
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
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
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4
5
6
7
8
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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
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
Editor's Notes
Persisters are dormant non –dividing variants of regular cells that form stochastically in microbial populations under the control of T/AT modules and are higly tolerant to antibiotics. However, unlike drug resistance, drug tolerance appear to be a transient end reversible physiological state in a small subpopulation of genetically identical cells. When the antimicrobial agent is removed, these persisting microbial cells not only resume growth, but their progeny is still susceptible to the antimicrobial agent.
Persisters are common to all bacterial species and play a pivotal role in microbial biofilm formation. Persisters are selected in patients with CF and similarly in patients with Candida oral thrush biofilm.
This observation suggest that persister may be responsible for the recalcitrance of chronic infections.
Le cellule persistenti sono varianti dormienti che non si dividono delle cellule normali che si formano stocasticamente in popolazioni microbiche sotto il controllo del sistema T / AT e sono altamente tolleranti agli antibiotici. Tuttavia, a differenza della resistenza ai farmaci, la tolleranza sembra essere uno stato fisiologico reversibile e transitoria presente in una piccola sottopopolazione di cellule geneticamente identiche. Quando l'agente antimicrobico viene rimosso, queste cellule microbiche persistenti riprendono la crescita, e la loro progenie risulta ancora suscettibile all'agente antimicrobico. Le cellule persistenti sono comuni a tutte le specie batteriche e svolgono un ruolo fondamentale nella formazione di biofilm microbico. Cellule persistenti sono state selezionate in pazienti con CF e similmente in pazienti con mughetto. Questa osservazione suggerisce che le cellule persistenti possono essere responsabili della recrudescenza delle infezioni croniche.
Looking to the bacterial lifecycle in lab curve pesisters cells form prevalently during the NON-GROWING stationary phase reaching a maximum of aproximately 1%
Guardando la classica curva di crescita batterica in vitro le cellule batteriche persistenti si formano prevalentemente nella fase stazionaria dove raggiungono un max di circa l’1%
Importantly
Persisters are not formed in response to antibiotic treatment
Da sottolineare il fatto che le cellule persistenti non si formano in risposta al trattamento antibiotico
There are more than one mechanism involved in persister cells formation, completely indipendent one another.
They are suggested to arise spontaneously (randomly) and continously during population growth (so-called type II or stochastic) or are formed in respose to an external (i.e. environmental) trigger (type I or induced).
A number of environmental signals can modulate the level of persistence in an isogenic microbial population.
I meccanismi per i quali si formano sono molteplici e indipendenti tra loro: possono sorgere spontaneamente (in maniera random) e continuamente durante la crescita cellulare (tipo II o stocastico) oppure formarsi in risposta ad uno stimolo esterno (per esempio ambientale) (tipo I o indotto).
Molteplici segnali ambientali possono modulare il livello di PC in una popolazione microbica:
There is increasing evidence that a significant environmental impact on bacteria is stress, which, in effecting a myriad of adaptative and protective responses, alters gene expression patterns and cell physiology in ways that can and do influence antimicrobial susceptibility. This occurs indirectly, as a result of stress-induced growth cessation or dormancy (tolerance), since antimicrobials act on growing cells, or directly as a result of the stress-dependent recruitment of resistance determinants. 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
C'è una crescente evidenza che un impatto ambientale significativo sui batteri è lo stress, che, nell’attivare una miriade di risposte di adattamento e protettive, altera gli schemi di espressione genica e fisiologica cellulare in modi che possono influenzare e influenzano la suscettibilità agli antibiotici. Questo avviene indirettamente, come conseguenza stress indotta della cessazione della crescita o dormienza (tolleranza), poiché antimicrobici agiscono sulle cellule in crescita, o direttamente in conseguenza del reclutamento stress-dipendente di determinanti di resistenza. K. Lewis e co-autori hanno scoperto, mediante analisi transcriptome, che in persisters ci sono overexpressed geni che sono presenti in tutti i cromosomi di batteri e chiamato tossina / antitossina
This system consists of two genes on the same operon, one encoding a stable TOX, typically a protein capable of inhibiting an essential cellular process as the translation or replication, and the other encoding a labile ATOX that prevents the action of the TOX forming together the inactive complex T / AT. When the cell is in the presence of an external factor potentially lethal "stress" we have the degradation of the AT thus the T no longer inhibited can perform its action leading to the arrest of the division or to the cell death.
Questo sistema è costituito da due geni sullo stesso operone che codificano uno una TOX stabile tipicamente una proteina capace di inibire un processo cellulare essenziale come la traslazione o la replicazione, e l’altro una ATOX labile che previene l’azione della TOX formando insieme il complesso inattivo T/AT. Quando la cellula è in presenza di un fattore esterno potenzialmente letale lo “stress” induce la disgregazione del AT di conseguenza la T non più inibita può svolgere la sua azione portando all’arresto della divisione o alla morte della cellula.
Persiter cells were discovered 70 yrs ago, yet we are only now beginnig to understand the mechanism of their formation.
In 1944, Joseph Bigger, a medical doctor from the university of Dublin, was working in a military hospital in York, experimenting with the recently introduced penicillin. Addition of penicillin to a culture of Staphylococcus resulted in lysis. Bigger plated this transparent liquid and recovered surviving colonies. Upon re inoculation, these colonies grew into a culture that again lysed in the presence of penicillin but formed a new sub-population of what bigger called “persister” to differentiate them from resistant mutants. He came to the conclusion that…..
Le PC sono state scoperte 70 anni fa, ma solo ora stiamo iniziando a capire il meccanismo della loro formazione. Nel 1944, Joseph Bigger, un medico presso l'università di Dublino, stava lavorando in un ospedale militare a York, sperimentando con la penicillina recentemente introdotta. L'aggiunta di penicillina ad una cultura di Staphylococcus porta a lisi. Bigger piastrò questo liquido trasparente e recuperò delle colonie sopravvissute. Se reinoculate, queste colonie crescono in una nuova cultura che ancora una volta si lisa in presenza di penicillina, ma forma una nuova sotto-popolazione di ciò che Bigger chiamò "persister" per differenziarli dai mutanti resistenti. Arrivò alla conclusione che .....
The bacterial resistance to antimicrobials is a genetic phenomenon for which a bacterium is insensitive to the activity of one or more antibiotics. It is a condition that involves the entire bacterial population. Generally, bacteria develop resistance through genetic modifications triggered by previous exposure of the pathogen to the antibiotic and may be accomplished by different mechanisms. decreased permeability increased number of efflux pumps modification of the target modification of the drug molecule with loss of activity
La resistenza batterica ai farmaci antimicrobici è il fenomeno su scala genetica per il quale un batterio risulta insensibile all’ attività di uno o più antibiotici. Si tratta di una condizione che coinvolge tutta la popolazione batterica. Generalmente i batteri sviluppano resistenza attraverso modificazioni genetiche scatenate da una precedente esposizione del patogeno all’antibiotico e può avvenire secondo diversi meccanismi.
diminuita permeabilità
aumentato numero delle pompe ad efflusso
modificazione del target
modificazione della molecola del farmaco con perdita dell’attività
Bacterial dormant or persister cells are only a small fraction of the bacterial population that is tolerant to the drug ... The paradox that emerges is that chronic infections are often supported by micro-organisms sensitive to the antibiotic at all even if the disease is difficult or impossible to eradicate.
Le cellule batteriche dormienti o persistenti sono invece solo una piccola frazione della popolazione batterica che risulta tollerante al farmaco…
Il paradosso che emerge è che le infezioni ricorrenti spesso risultano sostenute da microorganismi del tutto sensibili all’antibiotico anche se la patologia risulta difficile o impossibile da eradicare.
Tolerance works in a different way, not by preventing antibiotic binding but by interfering with the lethal action of antimicrobial agents
La tolleranza è diversa dalla resistenza: infatti non previene l’azione del farmaco ma ne impedisce l’azione letale.
The figure shows the difference between tolerance and resistance.
Normaly the antibiotic binds to the target altering its function, which causes cell death.
In the case of resistance 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.
In the case of tolerance a different molecule inhibits the antibiotic target. This prevents the antibiotic from corrupting its functions, resulting in tolerance
Questa figura aiuta a capire.
Normalmente l’agente antimicrobico si lega al suo target alterando la sua funzione e causando la morte cellulare…
Se il batterio è resistente il bersaglio può essere modificato impedendo all’antibiotico di legarsi
Nel caso della tolleranza il farmaco si lega al bersaglio ma una molecola diversa lo inibisce prevenendo l’azione dell’antibiotico.
The figure shows the treatment of a population with an antibiotic, wich resuls in cell death, leaving only persister cells or resistant mutants alive
Quindi in presenza di un antibiotico le cellule regolari muoiono, quelle resistenti crescono mentre le persistenti rimangono inalterate
When the concentration of the drug decreases the new population that originates from resistant cells will be formed by all resistant cells while the new population which is formed by persister cells will be formed by suseptible regular cells and by a new population of persister cells
Quando la concentrazione del farmaco diminuisce la nuova popolazione che origina da cellule resistenti sarà formata da tutte cellule resistenti mentre quella che si forma dalle cellule persistenti sarà formata da cellule regolari del tutto sensibili e da una nuova frazione di persistenti
This behavior explains what happens in chronic infections that recur after the end of antibiotic course while maintaining sensitivity to the drug itselff
Questo comportamento spiega l’andamento delle infezioni croniche che si ripresentano dopo che si è terminato il ciclo antibiotico mantenendo la sensibilità al farmaco.
According with CDC, 65% of all infections in developed countries are caused by biofilm. Biofilm can be formed by most, if not all pathogens. Biofilm infections are highly recalcitrant to antibiotic treatment. However, planktonic cells that are derived from these biofilm are, in most case, fully susceptible to antibiotics. Importantly, biofilm do not actually growth in the presence of elevated concentrations of antibiotics (concentrations required to kill biofilms are not therapeutically achievable), therefore biofilm do not have increased resistance compared with planktonic cells
Secondo il CDC, il 65% di tutte le infezioni nei paesi sviluppati sono causate da biofilm. Il biofilm può essere formato dalla maggior parte, se non tutti i microorganismi patogeni. Le infezioni da biofilm sono altamente recalcitranti al trattamento antibiotico. Tuttavia, le cellule planctoniche che derivano dai biofilm sono, nella maggior parte dei casi, completamente sensibili agli antibiotici. Importante, i biofilm effettivamente non crescono in presenza di elevate concentrazioni di antibiotici (ma le concentrazioni richieste per uccidere i biofilm non sono terapeuticamente raggiungibili), anche se i biofilm non hanno una maggiore resistenza rispetto alle cellule planctoniche
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.
Allora, come spiegare la resistenza agli agenti antimicrobici dei biofilm 100/1000 volte maggiore rispetto alle corrispondenti cellule flottanti se non sono presentano meccanismi di resistenza?
Il colpevole sono le cellule persistenti
Infatti, nei biofilm sono presenti le cellule persistenti….il microambiente è favorevole ma quando i nutrimenti vengono meno, specialmente negli strati profondi, lo stato dormiente diventa una opzione favorevole. La percentuale di PC si aggira tra lo 0.1 -1%.
Le PC sembrano essere la risposta alla tolleranza dei biofilm agli antibiotici.
Come fanno i biofilm a sopravvivere?
Inizialmente l’antibiotico uccide la maggior parte della popolazione batterica normale nel biofilm e plank, il sistema immunitari si occupa delle PC plank mentre non raggiunge e non elimina quelle nel BF protette dalla matrice che, non appena le condizioni tornano favorevoli resuscitano e l’infezione ritorna.
In addition the pC are a problem because they may represent a reservoir of microorganisms from which resistant mutants can arise. it‘s well known that responses to stress can promote both adaptive mutations and horizontal gene transfer between bacteria.
Oltre a questo le pC sono un problema perché possono rappresentare un serbatoio di microorganismi dal quale possono formarsi poi mutanti resistenti.
E’ noto infatti che risposte agli stress possono promuovere sia mutazioni adattative che il trasferimento orizzontale di geni tra batteri.
The PCs are present in both bacteria and fungi and can explain many chronic infections that require prolonged pharmacological treatments such as:
Le PC sono presenti sia nei batteri che nei funghi e possono spiegare molte infezioni croniche che richiedono trattamenti farmacologici prolungati come:
The question we ask is .... There are medications that can affect the persister cells? Currently not, being all the antimicrobial agents used in clinical practice linked to the viability of the bacteria to be active. The strategy to be followed for antipersisters drugs development must be different from that used for standard antibiotics based on the inhibition of a single vital mechanism. In fact in the case of PC ..... the mechanisms involved are many
La domanda che ci poniamo è….
Esistono farmaci che possono colpire la cellule persistenti? Attualmente no essendo tutto l’armamentario in uso nella pratica clinica basato sulla vitalità del batterio per poter funzionare. La strategia da seguire per il loro sviluppo quindi deve essere diversa da quella usata per un antibiotico standard basata sull’inibizione di un singolo meccanismo vitale…..nel caso delle PC i meccanismi implicati sono infatti molteplici
One strategy might be that suggested by Lewis to develop a PRO-antibiotic initialy not active but activated by cytoplasm enzymes and able to hit more targets at the same time killing the cell. like METRONIDAZOLE.
Una strategia potrebbe essere quella suggerita da Lewis di sviluppare un PRO-antibiotico non attivo di per se ma attivato da enzimi citoplasmatici e capace di colpire più bersagli uccidendo la cellula un po’ come fa il METRONIDAZOLO.
Photodynamic therapy may act as the “perfect antibiotic” but it’s hard to prove sperimentally because off PCs can not be cultured in vitro and their temporary phenotype precludes introducing them into an animal model of infection
La PDT potrebbe funzionare come il perfetto antibiotico suggerito da Lewis ma è difficile da provare sperimentalmente perché le cellule persistenti non possono essere coltivate in vitro e il loro fenotipo temporaneo fa si che non possono essere utilizzate neanche in modelli animali di infezione.