71 research outputs found
Corrigendum to “Performance of Candida albicans germ tube antibodies (CAGTA) and its association with (1 → 3)-β-D-glucan (BDG) for diagnosis of invasive candidiasis (IC)” (Diagnostic Microbiology & Infectious Disease (2019) 93(1) (39–43), (S073288931830230X), (10.1016/j.diagmicrobio.2018.07.007))
No full textIn the article “Performance of Candida albicans germ tube antibodies (CAGTA) and its association with (1 → 3)-β-D-glucan (BDG) for diagnosis of invasive candidiasis (IC),” the authors’ names are listed incorrectly. The correct names are as follows: Pietro Pini, Bruna Colombari, Enrico Marchi, Anna Castagnoli, Claudia Venturelli, Mario Sarti, Elisabetta Blasi
Candida metapsilosis as the least virulent member of the 'C. parapsilosis' complex
No full textResults of recent molecular studies have provided evidence of three distinct species within the Candida parapsilosis complex, namely Candida parapsilosis, Candida orthopsilosis and Candida metapsilosis. While there are initial data pertaining to the virulence of these Candida species with respect to reconstituted epidermal and oral epithelial tissues, there have been no studies, as of yet, on their interaction with immune cells. Employing an in vitro infection model using microglial cells, we investigated the pathogenetic potential of different isolates of each of these three species. We show that C. metapsilosis isolates are more susceptible to microglia-mediated antifungal activity, as compared with those of C. parapsilosis and C. orthopsilosis. Interestingly, C. metapsilosis isolates are also phagocytosed to a lower extent, but the yeast-containing phagosomes exhibit the highest degree of acidification in comparison with the phagosomes containing C. parapsilosis or C. orthopsilosis. Furthermore, when assessing microglia secretory response to infection, comparable high levels of MIP-1α and little or no TNF-α production are observed with all of these Candida species. Finally, unlike C. metapsilosis infected cells, microglial cells infected with C. parapsilosis and C. orthopsilosis release high and time-dependent levels of lactate dehydrogenase (LDH). Overall, these findings point to C. metapsilosis as the least virulent member of the ‘C. parapsilosis’ complex
Effects of Cupral® on the formation and persistence of microbial biofilms in vitro
No full textIntroduction: endodontic biofilm is a microbial community, enclosed in a polymeric matrix of polysaccharide origin where are frequently found pathogenic microorganisms, such as Gram+, Gram- and opportunistic fungi, belonging to Candida spp, responsible for several endodontic pathologies. As clinical importance is the fact that biofilm is extremely resistant to common intra-canal irrigants, antimicrobial drugs and host immune defenses. The aim of this in vitro study was to evaluate the efficacy of Cupral® on planktonic forms of some pathogens, as well as to assess its ability to prevent and affect the formation/persistence of microbial biofilms.
Materials and Methods: ATCC strains of S. aureus, P. aeruginosa and C. albicans were exposed to various concentrations of Cupral® (an antiseptic compound based on calcium and copper hydroxide, used in endodoncy) to investigate its antimicrobial efficacy. This activity has been evaluated in terms of microbial growth and cellular doubling time (optical density, colony forming units and doubling time assays), inhibition/persistence (crystal violet staining), viability of microbial cells embedded in the biofilms (live/dead stain) and pyoverdine production (fluorimetric assay). Finally, the morphology of Cupral®-treated biofilms was investigated by optical/confocal microscopy analysis.
Results: the addition of Cupral® to microbial cultures, influences, in a significantly and dose-dependent manner, the doubling time and growth of microbial cultures. Cupral® antimicrobial activity was also assessed on biofilms formation and persistence with meaningful decreases of residual biomass (observed reductions of 47-94% for S. aureus, 28-95% for P. aeruginosa and 27-75 % for C. albicans). Cupral®-treated biofilms analyzed by optical and confocal microscopy revealed loss of typical sessile structure, with few scattered microbial cells and a reduced thickness. Finally, the addition of Cupral® reduced both the number of embedded alive cells in the biofilms and the levels of pyoverdine in the culture supernatants.
Discussion and Conclusions: this pilot in vitro study provided the first evidences on Cupral® efficacy against microbial biofilms. The wide range of action (vs Gram+, Gram- and fungi) of Cupral® strongly suggests its use as compound in the prevention and treatment of main oral biofilm-associated infections
Nramp1 gene affects selective early steps in macrophage-mediated anti-cryptococcal defense
No full textCryptococcus neoformans is an opportunistic fungus responsible for severe and often recurrent meningoencephalitis in immunodepressed patients. Initial evidence suggests that C. neoformans is a facultative intracellular pathogen; however, the strategies by which C. neoformans undergoes survival and eventually proliferation have not been elucidated. We investigated the role of Nrampl gene in macrophage-mediated anti-cryptococcal defense. Using cell lines expressing the functional, mutated or knockout gene, it was established that Nramp1 (1) is not involved in the phagocytic event, (2) influences anti-cryptococcal activity in the early steps but not at later times, and (3) is unrelated to the biomolecular pathways through which C. neoformans impairs macrophage secretory response. Although the functional role of Nramp1 is still far from being elucidated, the present data add insight into its involvement in macrophage-mediated antimicrobial defense, particularly in the initial steps allowing C. neoformans growth inhibitio
Performance of Candida albicans germ tube antibodies (CAGTA) and its association with (1 → 3)-β-D-glucan (BDG) for diagnosis of invasive candidiasis (IC)
No full textInvasive candidiasis (IC) plays an important role as severe infection. Elder population, immunocompromised individuals, and intensive care unit (ICU) patients, especially when exposed to major surgery, are the most affected. IC diagnosis and treatment are difficult because of the absence of pathognomonic signs and symptoms. In addition, culture-based examination (gold standard) is known to have low sensitivity and long time to report. All these often lead to unnecessary and costly empirical antifungal therapies, burdened also by the onset of drug resistance and serious side effects for the patient. To partially overcome these problems, in recent years, novel noncultural markers have been investigated with the aim of easily and rapidly achieving an early diagnosis of IC. Such novel markers include the pan-fungal antigen (1 → 3)-β-D-glucan (BDG) and the anti–Candida albicans germ tube antibodies (CAGTAs). We retrospectively analyzed the presence of CAGTA on −80 °C stored serum samples, where the level of BDG had been previously assessed in a prospective study conducted in the Azienda Ospedaliero–Universitaria Policlinic of Modena (Pini et al. Infection 44:223–233, 2016). In particular, we selected 29 samples from proven IC episodes and 28 from non-IC cases. The 29 IC samples had been diagnosed as infections by C. albicans (n = 16), C. glabrata (n = 8), C. parapsilosis (n = 1), C. pelliculosa (n = 1), and C. tropicalis (n = 1), while 2 samples had intrasurgery biopsies positive for yeast (compatible with Candida spp.). The 28 control samples (non-IC) included 9 sera with positive blood cultures [E. faecium (n = 5), S. pneumoniae (n = 2), P. aeruginosa + A. baumannii (n = 2)] and 19 negative blood cultures. The CAGTA immunofluorescence assay was performed using 1:40, 1:80, 1:160, and 1:320 dilutions (reference dilution, as indicated by the manufacturer). According to the protocol, the samples were evaluated by the operator-dependent optical reading based on immunofluorescence positive/negative samples. In parallel, with the aim of standardizing the reading, the fluorescence images were captured, and the data were expressed as arbitrary fluorescence units (AFU). Finally, the results were interpreted as positive or negative using a cutoff provided by receiver operating characteristic (ROC) curves (Youden index). The traditional operator-dependent optical reading and the AFU measuring protocol provided comparable information with respect to the processed samples since IC and non-IC sera were correctly identified by the 2 CAGTA reading strategies in most of the cases. Interestingly, the AFU reading enabled a semiquantitative evaluation of the samples and an objective interpretation of the results. Based on the cutoff value, the AFU-based CAGTA procedure demonstrated a sensitivity of 52% and a specificity of 89%, while BDG showed a sensitivity of 90% and a specificity of 75%; the overall accuracy was 70% and 83% for CAGTA and BDG, respectively. The association of the 2 markers greatly increased both sensitivity and accuracy to 97% and 84%, respectively. As expected, when excluding non–C. albicans episodes, the sensitivity of CAGTA increased from 52% to 86%; moreover, with the exclusion of the non–deep-seated episodes, the sensitivity of CAGTA increased to 67% and reached 100% for C. albicans deep-seated candidiasis. Finally, when evaluating the influence of colonization, BDG demonstrated the most drastic decrease in specificity that dropped from 88% in noncolonized to 58% in colonized patients. With the exception of non–C. albicans episodes, CAGTA is a good marker of IC, particularly in the presence of deep-seated candidiasis. The performance of CAGTA greatly increases when used in combination with BDG
RUOLO DI PRODOTTI GENICI IDENTIFICATI MEDIANTE LIBRERIE LAMBDA-DISPLAY DA GENOMA COMPLETO DI PNEUMOCOCCO NELL’INTERAZIONE IN VITRO CON CELLULE MICROGLIALI ED EPITELIALI
No full textStreptococcus pneumoniae è un batterio responsabile di polmoniti, batteriemie, otiti medie e meningiti. In questo studio, mediante l’uso dei mutanti isogenici Δspr0075, Δspr1370 e Δspr1875 del ceppo acapsulato DP1004, abbiamo valutato il ruolo di queste proteine nell’interazione tra pneumococco e cellule microgliali ed epiteliali. Lo screening iniziale di una libreria genomica phage-display di S. pneumoniae con sieri di pazienti infettati ci ha permesso di identificare cloni fagici che portano epitopi B diversi. Tra questi sono stati isolati 6 frammenti antigenici corrispondenti a 3 proteine, mai identificate finora, codificate dalle ORFs spr0075, spr1370 e spr1875 della sequenza genomica del ceppo R6. Mediante modello di infezione in vitro abbiamo saggiato la suscettibilità dei suddetti mutanti alla fagocitosi, e la loro sopravvivenza all’interno delle cellule microgliali BV2. I risultati ottenuti dimostrano che tutti i ceppi sono efficientemente internalizzati; in particolare, i livelli di fagocitosi di Δspr0075 sono più bassi di quelli osservati con gli altri ceppi. Inoltre, la sopravvivenza dei mutanti all’interno della microglia è significativamente diversa: le CFU residue dei ceppi Δspr1370 e Δspr1875 sono rispettivamente più elevate e più basse di quelle osservate con Δspr0075 e DP1004. Mediante indagini al microscopio è stata infine valutata l’interazione dei diversi mutanti di pneumococco con le cellule epiteliali umane BEAS-2B, A549 e HEp-2. In tutti i casi si osserva una significativa riduzione della capacità di aderire alle cellule epiteliali in questione. Nel complesso, questi risultati indicano che le proteine identificate sono coinvolte nell’interazione di pneumococco con la microglia e le cellule epiteliali. In particolare, la proteina codificata dall’ORF spr1875 sembra svolgere un ruolo importante nella virulenza del batterio, in quanto il mutante Δspr1875 è più suscettibile del DP1004 all’attività antimicrobica microglia-mediata; tali evidenze sono in accordo con i risultati ottenuti in un modello murino, in cui il mutante capsulato Δspr1875 risulta essere meno virulento del ceppo wt D39 (Beninati C. et al., XVIII Lancefield International Symposium 2011)
Adaptive response of microglial cells to in vitro infection by Candida albicans isolates with different genomic backgrounds
No full textIt has been recently demonstrated that Candida albicans isolates with distinct genomic backgrounds (namely, b and c genotypes) press different susceptibility to antifungal activity by human monocytes in vitro. We show here that, although comparable in their ability to undergo dimorphic transition and in susceptibility to phagocytosis by microglial cells, the b and c isolates show striking differences in terms of intracellular survival. Only the c genotype resists indeed to intracellular killing and eventually replicates inside microglial cells, that in turn respond to fungal infection, preferentially towards the c genotype, with nuclear factor-kappa B (NF-kappa B) activation and increased Mip1 alpha production. These data indicate that C albicans-microglial cell interaction is strictly dependent upon fungal genotype, strengthening the potential significance of genotyping as prognostic parameter in clinical infections by C albicans. (c) 2006 Elsevier Ltd. All rights reserved
NF-kB activation and p38 phosphorilation in microglial cells infected with Leptospira or exposed to partially purified leptospiral lipoproteins
No full textRecently, we have shown a differential susceptibility of non-pathogenic vs. pathogenic leptospires to phagocytosis and killing by microglial cells. Although all ingested to some extent, only the pathogenic strains survived intracellularly while the non-pathogenic ones were killed in a time-dependent manner. By the same infection model, here we demonstrate that microglial cells respond to Leptospira infection with a time- and dose-dependent induction of molecular signals (p38 phosphorilation and NF-kB activation) and the production of soluble factors (cytokines and nitric oxide). Such bio-molecular response is predominantly observed against the pathogenic Leptospira; the phenomenon is reproduced by leptospiral lipoproteins and, to a lower extent, by leptospiral-derived LPS. These data provide initial evidence that Leptospira affects microglial cell response in a different manner depending upon the virulence of the infecting strain; specific bacterial components happen to be involved in the induction of such pathogen-induced immune response
POLIMORFISMO DEL GENE HWP1 IN CANDIDA ALBICANS: POSSIBILE RUOLO NELLA PRODUZIONE DI BIOFILM E NELL’INTERAZIONE CON IL MACROFAGO
No full textIntroduzione: Candida albicans (Ca) è frequentemente responsabile di candidosi profonde, soprattutto nei portatori di cateteri od altri
dispositivi medici, sulla cui superficie spesso si riscontra la comparsa di biofilm (1). La formazione di biofilm, a cui si associa la maggior
resistenza di Ca ai farmaci antifungini così come l’aumentata virulenza (2), procede secondo una complessa serie di eventi, finemente
controllati che coinvolgono numerosi geni tra cui HWP1 (Hyphal Wall Protein 1), codificante per una proteina essenziale per la crescita
ifale.
Il presente studio intende valutare il ruolo del biofilm ed in particolare del genopito HWP1 nell’interazione in vitro tra Ca e macrofago.
Materiali e metodi: isolati clinici di Ca recentemente identificati come eterozigoti ed omozigoti (3 per ciascun assetto genetico) per il
gene HWP1 (3), sono stati studiati in vitro per a) capacità di produrre biofilm, in presenza o assenza di siero fetale bovino e/o di CO2, e
b) suscettibilità alle cellule microgliali BV2 (4); le indagini hanno comportato la quantificazione della produzione di biofilm mediante
colorazione con cristal violetto o XTT, e valutazione della reattività del macrofago mediante dosaggio nei surnatanti di citochine, lattato
deidrogenasi, ecc.
Risultati: i ceppi saggiati hanno dimostrato a) aumentata produzione di biofilm in presenza di siero e di CO2 in tutti i ceppi, ma in modo
più evidente negli isolati omozigoti; b) maggiore resistenza verso la microglia, nel caso dei ceppi omozigoti per HWP1 e c) produzione
più elevata di TNFα in macrofagi esposti ai ceppi omozigoti.
Conclusioni: questi dati indicano che la produzione di biofilm conferisce a Ca una maggiore resistenza alla reattività della cellula
microgliale; la differenza osservata tra ceppi genotipicamente diversi per HWP1 suggerisce un ruolo importante di questo gene, oltre che
nella crescita delle ife, anche nella interazione con le cellule dell’immunità innata.
(1) Douglas, L. J. (2003). Trends Microbiol 11: 30–36
(2) Katragkou A et al. (2010). JID, 15:1941-1949
(3) Borghi et al. (2011). 21st ECCMID, Milano
(4) Blasi E et al. (1991). J Neuroimmunol 32: 49-5
IL GENOTIPO HWP1 DI CANDIDA ALBICANS INFLUENZA LA SUSCETTIBILITÀ FUNGINA ALLE CELLULE MICROGLIALI, MA NON LA PRODUZIONE DI BIOFILM
No full textIntroduzione. Il gene HWP1 di Candida albicans (Ca) codifica per una proteina della parete
cellulare fungina coinvolta nella crescita ifale e nell’adesione del lievito alle cellule epiteliali;
il ruolo di questa proteina nella patogenesi della candidosi non è ancora completamente
chiarito.
Il presente studio, mediante l’impiego di ceppi d’isolamento clinico di Ca e ceppi di
laboratorio isogenici per HWP1, intende valutare il ruolo del genotipo HWP1 nella
formazione del biofilm e nell’interazione in vitro tra Ca e le cellule microgliali (immunoeffettore
cerebrale).
Materiali e metodi. Sei ceppi clinici di Ca (3 eterozigoti e 3 omozigoti per il gene HWP1,
indicati rispettivamente con gli acronimi H/h e H/H) e 2 ceppi di laboratorio (DAY286, wildtype
per HWP1, H/H-wt, e FJS24, suo mutante isogenico HWP1-deleto, H/H-KO), sono stati
studiati in vitro per valutare: a) la capacità di formare biofilm, b) i livelli di espressione del
gene HWP1, c) l’ultrastruttura e d) la suscettibilità alle cellule microgliali, sia in termini di
inibizione della formazione del biofilm che di induzione di una risposta secretoria.
Risultati. Confrontando tra loro i ceppi clinici, i genotipi H/h hanno dimostrato una ridotta
capacità di formare biofilm e una maggiore suscettibilità al danno mediato dalla cellula
microgliale; a differenza della controparte H/H, i genotipi H/h hanno anche esibito una ridotta
capacità a stimolare la produzione di ossido nitrico e di TNFα da parte delle cellule
microgliali. A seguito dell’esposizione alle cellule microgliali, i ceppi H/H, ma non quelli
H/h, hanno mostrato aumentati livelli di mRNA HWP1 specifico.
Confrontando i 2 ceppi isogenici per HWP1, il genotipo H/H-KO ha mostrato una aumentata
suscettibilità al danno mediato dalla microglia rispetto al ceppo H/H-wt, mentre il biofilm è
stato prodotto in misura comparabile da parte di entrambi i ceppi. Inoltre, la microscopia
elettronica a trasmissione ha rivelato differenze tra i due ceppi in termini di spessore della
parete fungina, presenza di strutture superficiali adesine-simili e granuli intracitoplasmatici;
tali differenze sono mantenute anche in seguito all'esposizione alle cellule microgliali.
Conclusioni. Questi dati indicano che il genotipo HWP1 di Ca influenza la suscettibilità delle
cellule fungine alla microglia, ma non ha effetto sulla capacità del fungo di formare biofilm
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