Arianna Pompilio

Biofilm Formation by the Emerging Fungal Pathogen Trichosporon asahii: Development, Architecture, and Antifungal Resistance

ABSTRACT 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 μg/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.

Factors associated with adherence to and biofilm formation on polystyrene by Stenotrophomonas maltophilia: the role of cell surface hydrophobicity and motility

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.

Phenotypic and genotypic characterization of Stenotrophomonas maltophilia isolates from patients with cystic fibrosis: Genome diversity, biofilm formation, and virulence

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.

Antibacterial and anti-biofilm effects of cathelicidin peptides against pathogens isolated from cystic fibrosis patients

Six different cathelicidin-derived peptides were compared to tobramycin for antibacterial and anti-biofilm effects against S. aureus, P. aeruginosa, and S. maltophilia strains isolated from cystic fibrosis patients. Overall, SMAP-29, BMAP-28, and BMAP-27 showed relevant antibacterial activity (MIC(50) 4-8μg/ml), and in some cases higher than tobramycin. In contrast, indolicidin, LL-37, and Bac7(1-35) showed no significant antimicrobial activity (MIC(50)>32μg/ml). Killing kinetics experiments showed that in contrast to tobramycin the active cathelicidin peptides exert a rapid bactericidal activity regardless of the species tested. All three peptides significantly reduced biofilm formation by S. maltophilia and P. aeruginosa strains at 1/2× MIC, although at a lower extent than tobramycin. In addition, BMAP-28, as well as tobramycin, was also active against S. aureus biofilm formation. Preformed biofilms were significantly affected by bactericidal SMAP-29, BMAP-27 and BMAP-28 concentrations, although at a lesser extent than tobramycin. Overall, our results indicate the potential of some cathelicidin-derived peptides for the development of novel therapeutic agents for cystic fibrosis lung disease.

Comparative evaluation of the Vitek-2 Compact and Phoenix systems for rapid identification and antibiotic susceptibility testing directly from blood cultures of Gram-negative and Gram-positive isolates.

We performed a comparative evaluation of the Vitek-2 Compact and Phoenix systems for direct identification and antimicrobial susceptibility testing (AST) from positive blood culture bottles in comparison to the standard methods. Overall, 139 monomicrobial blood cultures, comprising 91 Gram-negative and 48 Gram-positive isolates, were studied. Altogether, 100% and 92.3% of the Gram-negative isolates and 75% and 43.75% of the Gram-positive isolates showed concordant identification between the direct and the standard methods with Vitek and Phoenix, respectively. AST categorical agreements of 98.7% and 99% in Gram-negative and of 96.2% and 99.5% in Gram-positive isolates with Vitek and Phoenix, respectively, were observed. In conclusion, direct inoculation procedures for Gram-negative isolates showed an excellent performance with both automated systems, while for identification of Gram-positive isolates they proved to be less reliable, although Vitek provided acceptable results. This approach contributes to reducing the turnaround time to result of blood cultures, with a positive impact on patient care.

Adhesion to and biofilm formation on IB3-1 bronchial cells by Stenotrophomonas maltophilia isolates from cystic fibrosis patients.

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.

Potential novel therapeutic strategies in cystic fibrosis: antimicrobial and anti-biofilm activity of natural and designed α-helical peptides against Staphylococcus aureus, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia.

BackgroundTreatment of cystic fibrosis-associated lung infections is hampered by the presence of multi-drug resistant pathogens, many of which are also strong biofilm producers. Antimicrobial peptides, essential components of innate immunity in humans and animals, exhibit relevant in vitro antimicrobial activity although they tend not to select for resistant strains.ResultsThree α-helical antimicrobial peptides, BMAP-27 and BMAP-28 of bovine origin, and the artificial P19(9/B) peptide were tested, comparatively to Tobramycin, for their in vitro antibacterial and anti-biofilm activity against 15 Staphylococcus aureus, 25 Pseudomonas aeruginosa, and 27 Stenotrophomonas maltophilia strains from cystic fibrosis patients. All assays were carried out in physical-chemical experimental conditions simulating a cystic fibrosis lung. All peptides showed a potent and rapid bactericidal activity against most P. aeruginosa, S. maltophilia and S. aureus strains tested, at levels generally higher than those exhibited by Tobramycin and significantly reduced biofilm formation of all the bacterial species tested, although less effectively than Tobramycin did. On the contrary, the viability-reducing activity of antimicrobial peptides against preformed P. aeruginosa biofilms was comparable to and, in some cases, higher than that showed by Tobramycin.ConclusionsThe activity shown by α-helical peptides against planktonic and biofilm cells makes them promising “lead compounds” for future development of novel drugs for therapeutic treatment of cystic fibrosis lung disease.

Effect of Environmental Factors on Biofilm Formation by Clinical Stenotrophomonas maltophilia Isolates

The influence of environmental factors (temperature, aerobiosis-anaerobiosis, static-dynamic conditions, pH) was determined on biofilm formation by 51S. maltophilia clinical isolates. The strains produced more biofilm at 32 °C than at 37 or 18 °C. Aerobic and 6 % CO2 atmosphere yielded comparable biofilm amounts, higher than under anaerobic conditions. Biofilm production was not affected by staticvs. agitated culture conditions. Biofilm production at pH 7.5 and 8.5 was comparable but significantly higher than at pH 5.5. The capacity of individual strains to form biofilm and thus contribute to the severity of some diseases is influenced by host traits and environmental conditions at the site of infection, and play an important role in the pathogenesis of biomaterial-related disease caused byS. maltophilia.

Role of Excessive Inflammatory Response to Stenotrophomonas maltophilia Lung Infection in DBA/2 Mice and Implications for Cystic Fibrosis

ABSTRACT Stenotrophomonas maltophilia is a pathogen that causes infections mainly in immunocompromised patients. Despite increased S. maltophilia isolation from respiratory specimens of patients with cystic fibrosis (CF), the real contribution of the microorganism to CF pathogenesis still needs to be clarified. The aim of the present study was to evaluate the pathogenic role of S. maltophilia in CF patients by using a model of acute respiratory infection in DBA/2 mice following a single exposure to aerosolized bacteria. The pulmonary bacterial load was stable until day 3 and then decreased significantly from day 3 through day 14, when the bacterial load became undetectable in all infected mice. Infection disseminated in most mice, although at a very low level. Severe effects (swollen lungs, large atelectasis, pleural adhesion, and hemorrhages) of lung pathology were observed on days 3, 7, and 14. The clearance of S. maltophilia observed in DBA/2 mouse lungs was clearly associated with an early and intense bronchial and alveolar inflammatory response, which is mediated primarily by neutrophils. Significantly higher levels of interleukin-1β (IL-1β), IL-6, IL-12, gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), GROα/KC, MCP-1/JE, MCP-5, macrophage inflammatory protein 1α (MIP-1α), MIP-2, and TARC were observed in infected mice on day 1 with respect to controls. Excessive pulmonary infection and inflammation caused systemic effects, manifested by weight loss, and finally caused a high mortality rate. Taken together, our results show that S. maltophilia is not just a bystander in CF patients but has the potential to contribute to the inflammatory process that compromises respiratory function.

Subinhibitory concentrations of moxifloxacin decrease adhesion and biofilm formation of Stenotrophomonas maltophilia from cystic fibrosis

Stenotrophomonas maltophilia is an emerging nosocomial bacterial pathogen that is currently isolated with increasing frequency from the airways of cystic fibrosis (CF) patients. In this study the effect of subinhibitory concentrations (subMICs) of moxifloxacin on adhesion, biofilm formation and cell-surface hydrophobicity of two strains of S. maltophilia isolated from CF patients were evaluated. Adhesion and biofilm formation assays were carried out on polystyrene and quantified by colony counts. Cell-surface hydrophobicity was determined by a test for adhesion to n-hexadecane. Moxifloxacin at 0.03x and 0.06x MIC caused a significant decrease in adhesion and biofilm formation by both strains tested. A significant reduction in cell-surface hydrophobicity following exposure to subMICs of moxifloxacin was observed for one strain only. The results of the present study provide an additional rationale for the use of moxifloxacin in CF patients and more generally in biofilm-related infections involving S. maltophilia.