Trichosporon asahii is the most common cause of fatal disseminated trichosporonosis, frequently associated with indwelling medical devices. Despite the use of antifungal drugs to treat trichosporonosis, infection is often persistent and is associated with high mortality. This drove our interest in evaluating the capability of T. asahii to form a biofilm on biomaterial-representative polystyrene surfaces through the development and optimization of a reproducible T. asahii-associated biofilm model. Time course analyses of viable counts and a formazan salt reduction assay, as well as microscopy studies, revealed that biofilm formation by T. asahii occurred in an organized fashion through four distinct developmental phases: initial adherence of yeast cells (0 to 2 h), germination and microcolony formation (2 to 4 h), filamentation (4 to 6 h), and proliferation and maturation (24 to 72 h). Scanning electron microscopy and confocal scanning laser microscopy revealed that mature T. asahii biofilms (72-h) displayed a complex, heterogeneous three-dimensional structure, consisting of a dense network of metabolically active yeast cells and hyphal elements completely embedded within exopolymeric material. Antifungal susceptibility testing demonstrated a remarkable rise in the MICs of sessile T. asahii cells against clinically used amphotericin B, caspofungin, voriconazole, and fluconazole compared to their planktonic counterparts. In particular, T. asahii biofilms were up to 16,000 times more resistant to voriconazole, the most active agent against planktonic cells (MIC, 0.06 g/ml). Our results suggest that the ability of T. asahii to form a biofilm may be a major factor in determining persistence of the infection in spite of in vitro susceptibility of clinical isolates.Candida species are the most common cause of disseminated nosocomial fungal infections (34). Invasive infections by rarer opportunistic fungal pathogens, however, have recently emerged as a significant problem in treatment of immunocompromised patients (8, 34).In particular, disseminated life-threatening Trichosporon infection is becoming increasingly common in patients with underlying hematological malignancies, extensive burns, solid tumors, and AIDS, accounting for approximately 10% of all confirmed cases of disseminated fungal infections (8,12,14,(32)(33)(34). Likewise, nonimmunosuppressed patients have suffered from Trichosporon infections associated with ophthalmologic surgery, infections of prosthetic devices, intravenous drug abuse, and peritoneal dialysis (1, 17, 21).Trichosporon beigelii was formerly considered the causative agent in trichosporonosis, but recent taxonomic findings based on partial sequences of large-subunit rRNA and DNA relatedness (9, 29, 30) revealed that T. beigelii actually consists of six distinct pathogenic Trichosporon species.In particular, Trichosporon asahii is the major cause of disseminated or deep-seated trichosporonosis (12,30). Although most of the reported cases of hematogenous T. asahii infections occurred in patie...

Helicobacter pylori persistence is associated with its capacity to develop biofilms as a response to changing environmental conditions and stress. Extracellular DNA (eDNA) is a component of H. pylori biofilm matrix but the lack of DNase I activity supports the hypothesis that eDNA might be protected by other extracellular polymeric substances (EPS) and/or Outer Membrane Vesicles (OMVs), which bleb from the bacteria surface during growth. The aim of the present study was to both identify the eDNA presence on OMVs segregated from H. pylori ATCC 43629/NCTC 11639 biofilm (bOMVs) and its planktonic phase (pOMVs) and to characterize the physical-chemical properties of the OMVs. The presence of eDNA in bOMVs and pOMVs was initially carried out using DNase I-gold complex labeling and Transmission Electron Microscope analysis (TEM). bOMVs and pOMVs were further isolated and physical-chemical characterization carried out using dynamic light scattering (DLS) analysis. eDNA associated with OMVs was detected and quantified using a PicoGreen spectrophotometer assay, while its extraction was performed with a DNA Kit. TEM images showed that eDNA was mainly associated with the OMV membrane surfaces; while PicoGreen staining showed a four-fold increase of dsDNA in bOMVs compared with pOMVs. The eDNA extracted from OMVs was visualized using gel electrophoresis. DLS analysis indicated that both planktonic and biofilm H. pylori phenotypes generated vesicles, with a broad distribution of sizes on the nanometer scale. The DLS aggregation assay suggested that eDNA may play a role in the aggregation of OMVs, in the biofilm phenotype. Moreover, the eDNA associated with vesicle membrane may impede DNase I activity on H. pylori biofilms. These results suggest that OMVs derived from the H. pylori biofilm phenotype may play a structural role by preventing eDNA degradation by nucleases, providing a bridging function between eDNA strands on OMV surfaces and promoting aggregation.

BackgroundStenotrophomonas maltophilia is emerging as one of the most frequently found bacteria in cystic fibrosis (CF) patients. In the present study, phenotypic and genotypic traits of a set of 98 isolates of S. maltophilia obtained from clinical (CF and non-CF patients) and environmental sources were comparatively evaluated.ResultsS. maltophilia exhibited a high level of genomic diversity in both CF and non-CF group, thus possibly allowing this bacterium to expand its pathogenic potentials. Strains sharing the same pulsotype infected different patients, thus likely indicating the occurrence of clonal spread or acquisition by a common source. CF isolates differed greatly in some phenotypic traits among each other and also when compared with non-CF isolates, demonstrating increased mean generation time and susceptibility to oxidative stress, but reduced ability in forming biofilm. Furthermore, in CF isolates flagella- and type IV pili-based motilities were critical for biofilm development, although not required for its initiation. Sequential isogenic strains isolated from the same CF patient displayed heterogeneity in biofilm and other phenotypic traits during the course of chronic infection. CF and non-CF isolates showed comparable virulence in a mouse model of lung infection.ConclusionsOverall, the phenotypic differences observed between CF and non-CF isolates may imply different selective conditions and persistence (adaptation) mechanisms in a hostile and heterogeneous environment such as CF lung. Molecular elucidation of these mechanisms will be essential to better understand the selective adaptation in CF airways in order to design improved strategies useful to counteract and eradicate S. maltophilia infection.

BackgroundStenotrophomonas maltophilia has recently gained considerable attention as an important emerging pathogen in cystic fibrosis (CF) patients. However, the role of this microorganism in the pathophysiology of CF lung disease remains largely unexplored. In the present study for the first time we assessed the ability of S. maltophilia CF isolates to adhere to and form biofilm in experimental infection experiments using the CF-derived bronchial epithelial IB3-1cell line. The role of flagella on the adhesiveness of S. maltophilia to IB3-1 cell monolayers was also assessed by using fliI mutant derivative strains.ResultsAll S. maltophilia CF isolates tested in the present study were able, although at different levels, to adhere to and form biofilm on IB3-1 cell monolayers. Scanning electron and confocal microscopy revealed S. maltophilia structures typical of biofilm formation on bronchial IB3-1 cells. The loss of flagella significantly (P < 0.001) decreased bacterial adhesiveness, if compared to that of their parental flagellated strains. S. maltophilia CF isolates were also able to invade IB3-1 cells, albeit at a very low level (internalization rate ranged from 0.01 to 4.94%). Pre-exposure of IB3-1 cells to P. aeruginosa PAO1 significantly increased S. maltophilia adhesiveness. Further, the presence of S. maltophilia negatively influenced P. aeruginosa PAO1 adhesiveness.ConclusionsThe main contribution of the present study is the finding that S. maltophilia is able to form biofilm on and invade CF-derived IB3-1 bronchial epithelial cells, thus posing a rationale for the persistence and the systemic spread of this opportunistic pathogen in CF patients. Experiments using in vivo models which more closely mimic CF pulmonary tissues will certainly be needed to validate the relevance of our results.

We tested 40 clinical Stenotrophomonas maltophilia strains to investigate the possible correlation between adherence to and formation of biofilm on polystyrene, and cell surface properties such as hydrophobicity and motility. Most of the strains were able to adhere and form biofilm, although striking differences were observed. Eleven (27.5%) of the strains were hydrophobic, with hydrophobicity greatly increasing as S. maltophilia attached to the substratum. A positive correlation was observed between hydrophobicity and levels of both adhesion and biofilm formation. Most of the isolates showed swimming and twitching motility. A highly significant negative correlation was observed between swimming motility and level of hydrophobicity. Hydrophobicity is thus a significant determinant of adhesion and biofilm formation on polystyrene surfaces in S. maltophilia.

Stenotrophomonas maltophilia has been recognized as an emerging multi-drug resistant opportunistic pathogen in cystic fibrosis (CF) patients. We report a comparative genomic and phenotypic analysis of 91 S. maltophilia strains from 10 CF patients over a 12-year period. Draft genome analyses included in silico Multi-Locus Sequence Typing (MLST), Single-Nucleotide Polymorphisms (SNPs), and pangenome characterization. Growth rate, biofilm formation, motility, mutation frequency, in vivo virulence, and in vitro antibiotic susceptibility were determined and compared with population structure over time. The population consisted of 20 different sequence types (STs), 11 of which are new ones. Pangenome and SNPs data showed that this population is composed of three major phylogenetic lineages. All patients were colonized by multiple STs, although most of them were found in a single patient and showed persistence over years. Only few phenotypes showed some correlation with population phylogenetic structure. Our results show that S. maltophilia adaptation to CF lung is associated with consistent genotypic and phenotypic heterogeneity. Stenotrophomonas maltophilia infecting multiple hosts likely experiences different selection pressures depending on the host environment. The poor genotype-phenotype correlation suggests the existence of complex regulatory mechanisms that need to be explored in order to better design therapeutic strategies.

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