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Persistercells
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.
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Persistercells
- 1. Persister bacterial cells Dr. GiulianiFrancesco 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 persistercell 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 thesecells 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 notformed 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 morethan 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 Thereis 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 Includingwhole population Genetically determined
- 10. A state inwhich 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 Toleranceworks 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 etal. (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 ofkilling 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 itis 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 Persistersare 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 biofilmavoid 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 responsesaccelerate 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 inHuman 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 inHuman 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 AntimicrobialTherapy “ 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 possibleto 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 mayact 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-workersdemonstrate 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 Bacterialgrowth in control plates as from literature Complete sterilization of the plates coming from the PDT treated sample
- 29.
- 30. Empty your mind. Beformless, 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
- #3 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.
- #4 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%
- #5 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
- #6 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:
- #7 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
- #8 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.
- #9 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 .....
- #10 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
- #11 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.
- #12 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.
- #13 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.
- #14 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
- #15 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
- #16 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.
- #17 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
- #18 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
- #19 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.
- #20 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.
- #21 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.
- #22 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:
- #24 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
- #25 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.
- #26 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.