In Vitro Newly Isolated Environmental Phage Activity against Biofilms Preformed by Pseudomonas aeruginosa from Patients with Cystic Fibrosis.

As disease worsens in patients with cystic fibrosis (CF), Pseudomonas aeruginosa (PA) colonizes the lungs, causing pulmonary failure and mortality. Progressively, PA forms typical biofilms, and antibiotic treatments determine multidrug-resistant (MDR) PA strains. To advance new therapies against MDR PA, research has reappraised bacteriophages (phages), viruses naturally infecting bacteria. Because few in vitro studies have tested phages on CF PA biofilms, general reliability remains unclear. This study aimed to test in vitro newly isolated environmental phage activity against PA isolates from patients with CF at Bambino Gesù Children's Hospital (OBG), Rome, Italy. After testing in vitro phage activities, we combined phages with amikacin, meropenem, and tobramycin against CF PA pre-formed biofilms. We also investigated new emerging morphotypes and bacterial regrowth. We obtained 22 newly isolated phages from various environments, including OBG. In about 94% of 32 CF PA isolates tested, these phages showed in vitro PA lysis. Despite poor efficacy against chronic CF PA, five selected-lytic-phages (Φ4_ZP1, Φ9_ZP2, Φ14_OBG, Φ17_OBG, and Φ19_OBG) showed wide host activity. The Φ4_ZP1-meropenem and Φ14_OBG-tobramycin combinations significantly reduced CF PA biofilms (p < 0.001). To advance potential combined phage-antibiotic therapy, we envisage further in vitro test combinations with newly isolated phages, including those from hospital environments, against CF PA biofilms from early and chronic infections.

Stenotrophomonas maltophilia mutant lacking flagella remains virulent in DBA/2N mice but is less efficient in stimulating TNF-α expression.

To understand the role of flagella of Stenotrophomonas maltophilia in lung infections, DBA/2N mice were challenged with aflagellate fliI- mutant and colonization, invasion and persistence, lung damage and inflammatory response compared, on days 1 and 3 post-exposure (p.e.), with that of the isogenic wild-type (wt). Following exposure to nebulized bacterial suspension, mice infected with wt and fliI- strains showed a comparable trend in body weight change, pulmonary persistence, lung damage and mortality rate over the study period considered. Interestingly, although on day 1 p.e. both strains colonized near all the spleens, on day 3 p.e. wt strain persisted in 40% of spleens, whereas fliI- mutant was completely cleared. No significant differences were found in MIP-2, IFN-γ and IL-6 pulmonary levels between groups over time, except for TNF-α whose levels on day 1 p.e. were significantly higher in mice infected with flagellated wt strain. Overall, our results indicate that in S. maltophilia flagella and motility might not represent virulence traits involved in the pathogenesis of lung infection. However, the evidence for a specific flagellar-induced TNF-α response warrants further study.

Phylogenetic relationships, biofilm formation, motility, antibiotic resistance and extended virulence genotypes among Escherichia coli strains from women with community-onset primitive acute pyelonephritis.

The present work set out to search for a virulence repertoire distinctive for Escherichia coli causing primitive acute pyelonephritis (APN). To this end, the virulence potential of 18 E. coli APN strains was genotypically and phenotypically assessed, comparatively with 19 strains causing recurrent cystitis (RC), and 16 clinically not significant (control, CO) strains. Most of the strains belong to phylogenetic group B1 (69.8%; p<0.01), and APN strains showed unique features, which are the presence of phylogroup A, and the absence of phylogroup B2 and non-typeable strains. Overall, the most dominant virulence factor genes (VFGs) were ecpA and fyuA (92.4 and 86.7%, respectively; p<0.05), and the mean number of VFGs was significantly higher in uropathogenic strains. Particularly, papAH and malX were exclusive for uropathogenic strains. APN and RC strains showed a significantly higher prevalence of fyuA, usp, and malX than of CO strains. Compared to RC strains, APN ones showed a higher prevalence of iha, but a lower prevalence of iroN, cnf1, and kpsMT-II. Hierarchical cluster analysis showed a higher proportion of two gene clusters (malX and usp, and fyuA and ecpA) were detected in the APN and RC groups than in CO, whereas iutA and iha clusters were detected more frequently in APN strains. The motility level did not differ among the study-groups and phylogroups considered, although a higher proportion of swarming strains was observed in APN strains. Antibiotic-resistance rates were generally low except for ampicillin (37.7%), and were not associated with specific study- or phylogenetic groups. APN and RC strains produced more biofilm than CO strains. In APN strains, iha was associated with higher biofilm biomass formation, whereas iroN and KpSMT-K1 were associated with a lower amount of biofilm biomass. Further work is needed to grasp the virulence and fitness mechanisms adopted by E. coli causing APN, and hence develop new therapeutic and prophylactic approaches.

Evolution of Stenotrophomonas maltophilia in Cystic Fibrosis Lung over Chronic Infection: A Genomic and Phenotypic Population Study.

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.

Stenotrophomonas maltophilia Phenotypic and Genotypic Diversity during a 10-year Colonization in the Lungs of a Cystic Fibrosis Patient.

The present study was carried out to understand the adaptive strategies developed by Stenotrophomonas maltophilia for chronic colonization of the cystic fibrosis (CF) lung. For this purpose, 13 temporally isolated strains from a single CF patient chronically infected over a 10-year period were systematically characterized for growth rate, biofilm formation, motility, mutation frequencies, antibiotic resistance, and pathogenicity. Pulsed-field gel electrophoresis (PFGE) showed over time the presence of two distinct groups, each consisting of two different pulsotypes. The pattern of evolution followed by S. maltophilia was dependent on pulsotype considered, with strains belonging to pulsotype 1.1 resulting to be the most adapted, being significantly changed in all traits considered. Generally, S. maltophilia adaptation to CF lung leads to increased growth rate and antibiotic resistance, whereas both in vivo and in vitro pathogenicity as well as biofilm formation were decreased. Overall, our results show for the first time that S. maltophilia can successfully adapt to a highly stressful environment such as CF lung by paying a "biological cost," as suggested by the presence of relevant genotypic and phenotypic heterogeneity within bacterial population. S. maltophilia populations are, therefore, significantly complex and dynamic being able to fluctuate rapidly under changing selective pressures.

Identification, antimicrobial resistance and molecular characterization of the human emerging pathogen Streptococcus gallolyticus subsp. pasteurianus.

This study aimed to retrospectively identify 22Streptococcus bovis clinical strains based on the new taxonomy, as well as to investigate their antibiotic-resistance and clonality. Strains were identified by Phoenix100 system, 16S rRNA sequencing, and two MALDI-TOF MS platforms (Bruker Biotyper, Vitek MS). Antibiotic resistance was determined both phenotypically and genotypically, and clonality was assessed by PFGE. Most of strains (63.6%) were isolated from urine, and diabetes was the most common underlying disease (31.8%). Phoenix100 system revealed all strains belonged to biotype II, and 16S rRNA sequencing identified all strains as S. gallolyticus subsp pasteurianus (SGSP). Although both MALDI-TOF MS systems correctly identified isolates to the species level, only Bruker Biotyper accurately identified to the subspecies level. Erythromycin-resistant strains (31.8%) were also clindamycin-resistant and positive for erm(B). Strains resistant to tetracycline (68.2%) were also resistant to erythromycin. PFGE showed high genetic variability identifying 17 different pulsotypes, most of which single.

Evaluation of antibacterial and antibiofilm mechanisms by usnic acid against methicillin-resistant Staphylococcus aureus.

AIM: To evaluate the antibacterial and antibiofilm mechanisms of usnic acid (USN) against methicillin-resistant Staphylococcus aureus from cystic fibrosis patients. MATERIALS & METHODS: The effects exerted by USN at subinhibitory concentrations on S. aureus Sa3 strain was evaluated by proteomic, real-time PCR and electron microscopy analyses. RESULTS & CONCLUSION: Proteomic analysis showed that USN caused damage in peptidoglycan synthesis, as confirmed by microscopy. Real-time PCR analysis showed that antibiofilm activity of USN is mainly due to impaired adhesion to the host matrix binding proteins, and decreasing lipase and thermonuclease expression. Our data show that USN exerts anti-staphylococcal effects through multitarget inhibitory effects, thus confirming the rationale for considering it 'lead compound' for the treatment of cystic fibrosis infections.

In vitro and in vivo evaluation of BMAP-derived peptides for the treatment of cystic fibrosis-related pulmonary infections.

Patients with cystic fibrosis require pharmacological treatment against chronic lung infections. The alpha-helical antimicrobial peptides BMAP-27 and BMAP-28 have shown to be highly active in vitro against planktonic and sessile forms of multidrug-resistant Pseudomonas aeruginosa, Staphylococcus aureus, and Stenotrophomonas maltophilia cystic fibrosis strains. To develop small antibacterial peptides for therapeutic use, we tested shortened/modified BMAP fragments, and selected the one with the highest in vitro antibacterial activity and lowest in vivo acute pulmonary toxicity. All the new peptides have shown to roughly maintain their antibacterial activity in vitro. The 1-18 N-terminal fragment of BMAP-27, showing MIC90 of 16 µg/ml against P. aeruginosa isolates and strain-dependent anti-biofilm effects, showed the lowest pulmonary toxicity in mice. However, when tested in a murine model of acute lung infection by P. aeruginosa, BMAP-27(1-18) did not show any curative effect. If exposed to murine broncho-alveolar lavage fluid BMAP-27(1-18) was degraded within 10 min, suggesting it is not stable in pulmonary environment, probably due to murine proteases. Our results indicate that shortened BMAP peptides could represent a starting point for antibacterial drugs, but they also indicate that they need a further optimization for effective in vivo use.

In vitro activity of levofloxacin against planktonic and biofilm Stenotrophomonas maltophilia lifestyles under conditions relevant to pulmonary infection in cystic fibrosis, and relationship with SmeDEF multidrug efflux pump expression.

The activity of levofloxacin against planktonic and biofilm Stenotrophomonas maltophilia cells and the role played by the multidrug efflux pump SmeDEF were evaluated under conditions relevant to the cystic fibrosis (CF) lung. MIC, MBC and MBEC of levofloxacin were assessed, against five CF strains, under 'standard' (CLSI-recommended) and 'CF-like' (pH 6.8, 5% CO2, in a synthetic CF sputum) conditions. Levofloxacin was tested against biofilms at concentrations (10, 50 and 100 µg mL(-1)) corresponding to achievable serum levels and sputum levels by aerosolisation. smeD expression was evaluated, under both conditions, in planktonic and biofilm cells by RT-PCR. The bactericidal effect of levofloxacin was decreased, in three out of five strains tested, under 'CF-like' conditions (MBC: 2-4 vs 8-16 µg mL(-1), under 'standard' and 'CF-like' conditions, respectively). Biofilm was intrinsically resistant to levofloxacin, regardless of conditions tested (MBECs ≥ 100 µg mL(-1) for all strains). Only under 'CF-like' conditions, smeD expression increased during planktonic-to-biofilm transition, and in biofilm cells compared to stationary planktonic cells. Our findings confirmed that S. maltophilia biofilm is intrinsically resistant to therapeutic concentrations of levofloxacin. Under conditions relevant to CF, smeD overexpression could contribute to levofloxacin resistance. Further studies are warranted to define the clinical relevance of our findings.

Cooperative pathogenicity in cystic fibrosis: Stenotrophomonas maltophilia modulates Pseudomonas aeruginosa virulence in mixed biofilm.

The present study was undertaken in order to understand more about the interaction occurring between S. maltophilia and P. aeruginosa, which are frequently co-isolated from CF airways. For this purpose, S. maltophilia RR7 and P. aeruginosa RR8 strains, co-isolated from the lung of a chronically infected CF patient during a pulmonary exacerbation episode, were evaluated for reciprocal effect during planktonic growth, adhesion and biofilm formation onto both polystyrene and CF bronchial cell monolayer, motility, as well as for gene expression in mixed biofilms. P. aeruginosa significantly affected S. maltophilia growth in both planktonic and biofilm cultures, due to an inhibitory activity probably requiring direct contact. Conversely, no effect was observed on P. aeruginosa by S. maltophilia. Compared with monocultures, the adhesiveness of P. aeruginosa on CFBE41o- cells was significantly reduced by S. maltophilia, which probably acts by reducing P. aeruginosa's swimming motility. An opposite trend was observed for biofilm formation, confirming the findings obtained using polystyrene. When grown in mixed biofilm with S. maltophilia, P. aeruginosa significantly over-expressed aprA, and algD-codifying for protease and alginate, respectively-while the quorum sensing related rhlR and lasI genes were down-regulated. The induced alginate expression by P. aeruginosa might be responsible for the protection of S. maltophilia against tobramycin activity we observed in mixed biofilms. Taken together, our results suggest that the existence of reciprocal interference of S. maltophilia and P. aeruginosa in CF lung is plausible. In particular, S. maltophilia might confer some selective "fitness advantage" to P. aeruginosa under the specific conditions of chronic infection or, alternatively, increase the virulence of P. aeruginosa thus leading to pulmonary exacerbation.

In vitro activity of colistin against biofilm by Pseudomonas aeruginosa is significantly improved under "cystic fibrosis-like" physicochemical conditions.

The impact of physicochemical conditions observed in cystic fibrosis (CF) lung on colistin activity against both planktonic and biofilm P. aeruginosa cells was evaluated. MIC, minimum bactericidal concentration (MBC), and minimum biofilm eradication concentration (MBEC) values were assessed against 12 CF strains both under "CF-like" (anaerobiosis, pH6.4) and "standard" (aerobiosis, pH7.4) conditions. The activity of colistin was significantly higher under "CF-like" conditions compared to "standard" ones, both against planktonic (MIC90: 1 and 4 µg/mL, respectively) and biofilm (MBEC90: 512 and 1.024 µg/mL, respectively) cells, as confirmed by scanning electron microscopy. Improved activity was not related to biofilm matrix amount. It may be necessary to adequately "rethink" the protocols used for in vitro assessment of colistin activity, by considering physicochemical and microbiological features in the CF lung at the site of infection. This could provide a more favorable therapeutic index, rationale for administration of lower doses, probably resulting in reduced toxicity and emergence of resistant clones.

New insights in Staphylococcus pseudintermedius pathogenicity: antibiotic-resistant biofilm formation by a human wound-associated strain.

BACKGROUND: Staphylococcus pseudintermedius is an opportunistic pathogen recognized as the leading cause of skin, ear, and post-operative bacterial infections in dogs and cats. Zoonotic infections have also recently been reported causing endocarditis, infection of surgical wounds, rhinosinusitis, and catheter-related bacteremia. The aim of the present study is to evaluate, for the first time, the pathogenic potential of S. pseudintermedius isolated from a human infection. To this end, strain DSM 25713, which was recently isolated from a wound of a leukemic patient who underwent a bone marrow transplantation, was investigated for biofilm formation and antibiotic-resistance under conditions relevant for wound infection. RESULTS: The effect of pH (5.5, 7.1, and 8.7) and the presence of serum (diluted at 1:2, 1:10, and 1:100) on biofilm formation was assessed through a crystal violet assay. The presence of serum significantly reduced the ability to form biofilm, regardless of the pH value tested. In vitro activity of eight antibiotics against biofilm formation and mature 48 h-old biofilms was comparatively assessed by crystal violet assay and viable cell count, respectively. Antibiotics at sub-inhibitory concentrations reduced biofilm formation in a dose-dependent manner, although cefoxitin was the most active, causing a significant reduction already at 1/8xMIC. Rifampicin showed the highest activity against preformed biofilms (MBEC90: 2xMIC). None of the antibiotics completely eradicated the preformed biofilms, regardless of tested concentrations. Confocal and electron microscopy analyses of mature biofilm revealed a complex "mushroom-like" architecture consisting of microcolonies embedded in a fibrillar extracellular matrix. CONCLUSIONS: For the first time, our results show that human wound-associated S. pseudintermedius is able to form inherently antibiotic-resistant biofilms, suggestive of its pathogenic potential, and consistent with recent reports of zoonotic infections.

Fatal sepsis by Klebsiella pneumoniae in a patient with systemic lupus erythematosus: the importance of postmortem microbiological examination for the ex post diagnosis of infection.

The utility of postmortem microbiology has continuously been a topic of controversy. The present study describes a case of fatal sepsis in a patient with systemic lupus erythematosus. Postmortem culture and genotyping analyses allowed us to identify Klebsiella pneumoniae as the cause of sepsis, revealing the inadequateness of antimicrobial therapy.

Exposure to extremely low-frequency magnetic field affects biofilm formation by cystic fibrosis pathogens.

AIMS: To evaluate the in vitro effects of extremely low-frequency magnetic field (ELF-MF) on growth and biofilm formation by Staphylococcus aureus, Pseudomonas aeruginosa, Burkholderia cepacia and Stenotrophomonas maltophilia strains from cystic fibrosis patients. MATERIALS & METHODS: The motion of selected ions (Fe, Ca, Cu, Zn, Mg, K, Na) was stimulated by the ion resonance effect, then influence on growth and biofilm formation/viability was assessed by spectrophotometry or viability count. RESULTS: Generally, exposure to ELF-MF significantly increased bacterial growth and affected both biofilm formation and viability, although with differences with regard to ions and species considered. CONCLUSION: Exposure to ELF-MF represents a possible new approach for treatment of biofilm-associated cystic fibrosis lung infections.

Stenotrophomonas maltophilia virulence and specific variations in trace elements during acute lung infection: implications in cystic fibrosis.

Metal ions are necessary for the proper functioning of the immune system, and, therefore, they might have a significant influence on the interaction between bacteria and host. Ionic dyshomeostasis has been recently observed also in cystic fibrosis (CF) patients, whose respiratory tract is frequently colonized by Stenotrophomonas maltophilia. For the first time, here we used an inductively mass spectrometry method to perform a spatial and temporal analysis of the pattern of changes in a broad range of major trace elements in response to pulmonary infection by S. maltophilia. To this, DBA/2 mouse lungs were comparatively infected by a CF strain and by an environmental one. Our results showed that pulmonary ionomic profile was significantly affected during infection. Infected mice showed increased lung levels of Mg, P, S, K, Zn, Se, and Rb. To the contrary, Mn, Fe, Co, and Cu levels resulted significantly decreased. Changes of element concentrations were correlated with pulmonary bacterial load and markers of inflammation, and occurred mostly on day 3 post-exposure, when severity of infection culminated. Interestingly, CF strain - significantly more virulent than the environmental one in our murine model - provoked a more significant impact in perturbing pulmonary metal homeostasis. Particularly, exposure to CF strain exclusively increased P and K levels, while decreased Fe and Mn ones. Overall, our data clearly indicate that S. maltophilia modulates pulmonary metal balance in a concerted and virulence-dependent manner highlighting the potential role of the element dyshomeostasis during the progression of S. maltophilia infection, probably exacerbating the harmful effects of the loss of CF transmembrane conductance regulator function. Further investigations are required to understand the biological significance of these alterations and to confirm they are specifically caused by S. maltophilia.

Antimicrobial and antibiofilm activity of secondary metabolites of lichens against methicillin-resistant Staphylococcus aureus strains from cystic fibrosis patients.

AIM: Three secondary metabolites of lichens - usnic acid, atranorin and fumarprotocetraric acid - were evaluated for their in vitro antibacterial and antibiofilm activities against three strains each of methicillin-susceptible and methicillin-resistant Staphylococcus aureus (MRSA) from cystic fibrosis patients. MATERIALS & METHODS: Antibacterial activity was assessed by broth microdilution, while antibiofilm activity was evaluated by spectrophotometry or viable count. RESULTS: Usnic acid was significantly more active than atranorin against planktonic cells, while fumarprotocetraric acid exhibited no activity. Atranorin was the most effective in counteracting adhesion to polystyrene, although usnic acid was more active against MRSA. Usnic acid and atranorin showed comparable activity against biofilm formation, although atranorin was more active against MRSA. Usnic acid was significantly more active than atranorin against preformed biofilms. CONCLUSION: Secondary metabolites of lichens may be considered to be 'lead compounds' for the development of novel molecules for the treatment of S. aureus infections in cystic fibrosis patients.

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.

BACKGROUND: Treatment 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. RESULTS: Three 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. CONCLUSIONS: The 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.

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

BACKGROUND: Stenotrophomonas 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. RESULTS: S. 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. CONCLUSIONS: Overall, 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.

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

BACKGROUND: Stenotrophomonas 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. RESULTS: All 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. CONCLUSIONS: The 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.