Staphylococcus aureus is a major human pathogen. Its toxins can cause disease of varying severity, including food poisoning and a number of infections. Several strains form biofilms, i.e. multicellular aggregates encased in an exopolysaccharide matrix (slime) produced by the bacteria themselves. Biofilm-producing staphylococci are frequently involved in infections associated with the use of medical devices (e.g. catheter-related bloodstream infections). These biofilms protect bacteria from both host defences and antibiotic therapy, resulting in considerable difficulty in eradication. Bacteria residing in biofilms need to cope with adverse environmental conditions, such as oxygen and nutrient deficiency, that can induce a “dormant” state, like the Viable But NonCulturable (VBNC) state. VBNC forms are a survival strategy of non-sporulating bacteria subjected to environmental stress (e.g. changes in temperature or visible light, oxygen or nutrient deficiency). The VBNC state, a potential reservoir of pathogenic microorganisms, has been described for several gram-positive and negative species, both environmental and clinical. This survival strategy is characterized by the absence of culturability on culture media, low metabolic activity, and morpho-functional changes. Moreover, optimal growth conditions can “resuscitate” true VBNC cells, restoring replication capacity and full metabolic activity. The ability of staphylococci to enter the VBNC state has never been demonstrated. This work aimed to establish (i) whether S. aureus biofilms can give rise to VBNC forms that can resuscitate in suitable environmental conditions, and (ii) the role of different stressors in inducing the VBNC state. Cultures of S. aureus 10850 (a strong slime producer) and S. aureus ATCC 25923 (a non-slime producer) were grown on filter membranes under conditions suitable for biofilm development (i.e. presence of glucose) and exposed to different stress conditions, i.e. depletion of nutrients, in the presence or absence of minimum inhibitory concentrations (MICs) and of 4-fold, 8-fold and 16-fold MICs of vancomycin, synercid and daptomycin, three antibiotics used to treat severe Gram-positive infections. Culturability was monitored at intervals of three days. After cells had reached the nonculturable state they were subjected to live-dead staining using SYBR Green I and propidium iodide and their viability was tested by epifluorescence microscopy and flow cytometry. Gene expression was analyzed using RT-PCR and Real-time RT-PCR assays targeting bacterial 16SrDNA, glsF (a housekeeping, species-specific gene) and nuc (the gene coding for a thermostable nuclease) genes. VBNC forms were then assessed for their ability to recover replication activity through exposure to favourable growth conditions. The results of these experiments showed that (i) S. aureus is capable of entering the VBNC state provided that cells are contained in biofilm, because viable but nonculturable cells of S. aureus ATCC 25923 (the non-slime producer) were never recovered; (ii) VBNC S. aureus forms can be induced by starvation and antibiotics; (iii) VBNC S. aureus cells retain their metabolic activity, as demonstrated by positive gene expression assays; and (iv) VBNC S. aureus cells exposed to suitable culture conditions can revert to the culturable state (resuscitation). VBNC staphylococci may have a significant clinical role, since they can pass from the dormant state, where they are undetectable by routine methods within biofilm, to full metabolic and replication activity, causing severe infections.
Staphylococcus aureus è uno dei più importanti patogeni umani. Può causare patologie di diversa gravità, tossinfezioni alimentari ed infezioni sistemiche. Molti ceppi sono capaci di sviluppare biofilm, cioè una matrice esopolisaccaridica (slime) adesa ad una superficie, all’interno della quale rimangono inglobati i microrganismi produttori. Gli stafilococchi produttori di biofilm sono frequentemente coinvolti nelle infezioni associate all’uso di dispositivi medici impiantabili, come cateteri venosi centrali. Tali infezioni risultano difficili da eradicare in quanto i batteri risultano protetti sia dai sistemi di difesa dell’ospite che dalla terapia antibiotica. All’interno del biofilm, al contempo, i microrganismi trovano condizioni ambientali sfavorevoli come la carenza di ossigeno e di nutrienti in grado di indurre la trasformazione in forme quiescenti, tra cui lo stato VBNC (Viable But NonCulturable). Le forme VBNC rappresentano una strategia di sopravvivenza delle cellule batteriche non sporulanti sottoposte a stress ambientali, come variazioni di temperatura, luce solare, concentrazione di ossigeno e carenza di nutrienti. Sono caratterizzate da assenza di coltivabilità nei classici terreni di coltura, attività metabolica ridotta, modificazioni morfo-funzionali e dalla capacità di recuperare la capacità replicativa (resuscitation), quando le condizioni tornano favorevoli. Sono state descritte per diverse specie, soprattutto ambientali, ed anche per patogeni umani, ma la possibilità per gli stafilococchi di entrare in uno stato VBNC non è stata mai dimostrata. Questa attività di ricerca è stata rivolta a valutare la possibilità che S. aureus possa entrare in uno stato vitale ma non coltivabile, capace di resuscitation, quando si trova all’interno di biofilm. A tale scopo sono stati allestiti modelli di biofilm in vitro del ceppo S. aureus 10850 (forte produttore di slime) e in parallelo di S. aureus ATCC 25923 (non produttore di slime) e sottoposti a condizioni di stress, in particolare carenza di nutrienti associata o meno alla presenza di vancomicina, synercid o daptomicina (antibiotici utilizzati per contrastare le infezioni gravi sostenute da S. aureus) in concentrazioni pari o superiori (4x, 8x, 16x) alla minima inibente (MIC). La presenza di cellule vitali nei campioni non più coltivabili è stata valutata mediante microscopia in epifluorescenza e citometria a flusso dopo colorazione live-dead con i fluorocromi SYBR Green I e Ioduro di propidio; la capacità di espressione genica delle forme VBNC rilevate è stata analizzata mediante saggi di RT-PCR e Real-time RT-PCR specifici per il 16SrDNA batterico e per i geni glsF e nuc, il primo housekeeping specie-specifico per S. aureus e il secondo codificante per un fattore di virulenza. E’ stata inoltre valutata la possibilità delle forme VBNC di recuperare l’attività replicativa (resuscitation) ricreando condizioni di crescita favorevoli. I risultati ottenuti hanno dimostrato che (i) S. aureus è in grado di entrare in uno stato VBNC, ma solo all’interno di biofilm. Infatti forme VBNC sono state ottenute solo con il ceppo produttore di biofilm S. aureus 10850, mentre non è stato possibile rilevare la presenza di cellule vitali ma non coltivabili di S. aureus ATCC 25923; ii) condizioni di starvation in presenza di basse concentrazioni di vancomicina, synercid o daptomicina possono indurre lo stato VBNC; (iii) nello stato non coltivabile le cellule di S. aureus mantengono un’attività metabolica, come dimostrato dall’espressione di tutti i geni analizzati e che (iv) le forme VBNC di S. aureus sono in grado di riacquisire la capacità replicativa se poste in presenza di terreni di coltura addizionati di metaboliti essenziali per lo sviluppo cellulare, come il piruvato di sodio o il filtrato di una brodocoltura del ceppo wild-type S. aureus 10850. Questi risultati indicano che le forme VBNC di stafilococco potrebbero avere un ruolo rilevante in patologia umana in quanto capaci di rimanere latenti, e non evidenziabili con i comuni test colturali, all’interno del biofilm, ed essere causa di infezioni ricorrenti in seguito al recupero della piena attività metabolica e della conseguente capacità di moltiplicarsi.
Induction of viable but nonculturable state in Staphylococcus aureus biofilms obtained in vitro / Pasquaroli, Sonia. - (2011 Feb 11).
Induction of viable but nonculturable state in Staphylococcus aureus biofilms obtained in vitro
Pasquaroli, Sonia
2011-02-11
Abstract
Staphylococcus aureus is a major human pathogen. Its toxins can cause disease of varying severity, including food poisoning and a number of infections. Several strains form biofilms, i.e. multicellular aggregates encased in an exopolysaccharide matrix (slime) produced by the bacteria themselves. Biofilm-producing staphylococci are frequently involved in infections associated with the use of medical devices (e.g. catheter-related bloodstream infections). These biofilms protect bacteria from both host defences and antibiotic therapy, resulting in considerable difficulty in eradication. Bacteria residing in biofilms need to cope with adverse environmental conditions, such as oxygen and nutrient deficiency, that can induce a “dormant” state, like the Viable But NonCulturable (VBNC) state. VBNC forms are a survival strategy of non-sporulating bacteria subjected to environmental stress (e.g. changes in temperature or visible light, oxygen or nutrient deficiency). The VBNC state, a potential reservoir of pathogenic microorganisms, has been described for several gram-positive and negative species, both environmental and clinical. This survival strategy is characterized by the absence of culturability on culture media, low metabolic activity, and morpho-functional changes. Moreover, optimal growth conditions can “resuscitate” true VBNC cells, restoring replication capacity and full metabolic activity. The ability of staphylococci to enter the VBNC state has never been demonstrated. This work aimed to establish (i) whether S. aureus biofilms can give rise to VBNC forms that can resuscitate in suitable environmental conditions, and (ii) the role of different stressors in inducing the VBNC state. Cultures of S. aureus 10850 (a strong slime producer) and S. aureus ATCC 25923 (a non-slime producer) were grown on filter membranes under conditions suitable for biofilm development (i.e. presence of glucose) and exposed to different stress conditions, i.e. depletion of nutrients, in the presence or absence of minimum inhibitory concentrations (MICs) and of 4-fold, 8-fold and 16-fold MICs of vancomycin, synercid and daptomycin, three antibiotics used to treat severe Gram-positive infections. Culturability was monitored at intervals of three days. After cells had reached the nonculturable state they were subjected to live-dead staining using SYBR Green I and propidium iodide and their viability was tested by epifluorescence microscopy and flow cytometry. Gene expression was analyzed using RT-PCR and Real-time RT-PCR assays targeting bacterial 16SrDNA, glsF (a housekeeping, species-specific gene) and nuc (the gene coding for a thermostable nuclease) genes. VBNC forms were then assessed for their ability to recover replication activity through exposure to favourable growth conditions. The results of these experiments showed that (i) S. aureus is capable of entering the VBNC state provided that cells are contained in biofilm, because viable but nonculturable cells of S. aureus ATCC 25923 (the non-slime producer) were never recovered; (ii) VBNC S. aureus forms can be induced by starvation and antibiotics; (iii) VBNC S. aureus cells retain their metabolic activity, as demonstrated by positive gene expression assays; and (iv) VBNC S. aureus cells exposed to suitable culture conditions can revert to the culturable state (resuscitation). VBNC staphylococci may have a significant clinical role, since they can pass from the dormant state, where they are undetectable by routine methods within biofilm, to full metabolic and replication activity, causing severe infections.File | Dimensione | Formato | |
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