Although recent clinical evidence highlights an increase in the frequency of isolation of S. maltophilia from respiratory tract of CF patients, the role of this microorganism in the pathophysiology of CF lung disease, as well as patient-to-patient spread, have not yet been clearly elucidated [5, 7–9]. Moreover, the correlation between S. maltophilia persistent lung colonization and reduced pulmonary function first reported by Karpati et al , has not yet been confirmed by further studies [23–26]. On the other hand, the increased isolation of S. maltophilia from the sputa of CF patients has become a cause of concern in the CF community, as the organism is highly resistant to many of the antibiotics prescribed in CF management .
Because of its increasing clinical relevance, its high level of antibiotic-resistance, and the paucity of information on its specific role in the pathogenesis of CF lung infections, new information regarding the interactions between S. maltophilia and CF airway tissues are of paramount importance. To our knowledge, this is the first study which evaluated the ability of CF-derived S. maltophilia clinical isolates to adhere to and form biofilm in experimental infection experiments using the CF-derived bronchial epithelial IB3-1cell line.
Employing an in vitro static culture model, by using electron and confocal microscopy and determining the number (cfu) of attached bacteria at different time points post-infection, we showed that all the twelve studied CF-derived S. maltophilia isolates were able, although at different levels, to adhere and form biofilm when co-cultured with IB3-1 cell monolayers. Such results suggest that these characteristics might be highly conserved among S. maltophilia strains isolated from CF patients. Electron and confocal microscopy revealed S. maltophilia structures typical of biofilm formation on almost all bronchial IB3-1 cells. In particular, the overall cellular areas occupied by bacteria and their numbers are suggestive of the formation of microcolony, a finding reminiscent of the "flat" biofilm phenotype produced by P. aeruginosa, significantly different from the "mushroom-like" phenotype . Electron microscopy photographs revealed that S. maltophilia adhered to IB3-1 cells loses its cell profile, probably due to the presence of extracellular matrix. In fact, CLSM examination showed microcolony embedded in extracellular matrix whose production was significantly increased following exposure to S. maltophilia.
The ability of S. maltophilia to form biofilm on IB3-1 cells may contribute to explain why S. maltophilia tends to produce persistent infections in chronic obstructive pulmonary disease despite intensive antibiotic treatment . In this context, it is worth mentioning that some antibiotic-resistant S. maltophilia strains may persist in CF patients pulmonary tissue for up to 3 years, and that many patients are colonized at the same time with multiple strains of S. maltophilia .
Invasion of epithelial respiratory cells has been reported for CF-derived S. maltophilia clinical isolates [10, 20]. We have recently reported that, with the exception of an environmental S. maltophilia isolate (strain LMG959) all the CF-derived strains assayed were able to invade A549 cells . In the present study we evaluated the ability of twelve S. maltophilia CF isolates to invade IB3-1 cells, by classical invasion assays. The results obtained clearly indicated, for the first time, that S. maltophilia CF isolates were able to invade IB3-1 cells, albeit at a very low level (data not shown). Since strains presented a significant degree of heterogeneity in internalization efficiencies, it might be possible to hypothesize that S. maltophilia entry within IB3-1 cells may be strain-dependent. Together with the ability to form biofilm, the capability of S. maltophilia to enter IB3-1 might also explain the tendency of this microorganism to become persistent within CF pulmonary tissues, since within intracellular compartments it could find protection against host defenses and the reach of antibiotics. Moreover, internalization may likely influence the modulation of the inflammatory response of the infected host.
It has been reported that flagella could act as adhesins which play a role in bacterial binding to host mucosal surfaces as well as to abiotic surfaces [22, 31]. To study the role of flagella in the adhesiveness of S. maltophilia, we generated two independent mutants presenting a deletion encompassing the fliI gene of S. maltophilia strains OBGTC9 and OBGTC10. fliI encodes a substrate-specific ATPase (FliI), an enzyme necessary to provide energy for the export of flagellar structural components in a wide range of bacterial species . Swimming ability of the two mutant strains was almost completely abolished (Figure 4B). When co-cultured with IB3-1 cell monolayers, the two mutants showed a reduced capacity to adhere to IB3-1 cells, if compared to that of parental wild type strains (Figure 4A). Further, we showed that neither swimming nor twitching motilities were significantly associated to adhesion to or biofilm formation on IB3-1 cells. Thus, taken together, our results suggest that although flagella must play some role in S. maltophilia adhesiveness, regardless of their functionality, other structures must also be involved in this phenomenon, since the fliI mutation only attenuates, but not abolishes, the ability of S. maltophilia strains to adhere to IB3-1 cells.
We were not able to assess the role of flagella in S. maltophilia biofilm formation since exposure of IB3-1 monolayers to fliI
- mutant strains caused their disruption already after 6h-exposure. Further investigations are warranted to explain this apparent increased virulence following the loss of motility.
A number of methods have been developed for cultivation and quantification of biofilms , but no standardized protocol for assessment of biofilm formation has been established so far. Nevertheless, the microtiter plate method remains among the most frequently used assays for investigation of biofilm formation, and a number of modifications have been developed for the cultivation and quantification of bacterial biofilms . Since S. maltophilia biofilm formation on abiotic surfaces is generally considered less relevant than biofilm formation on cultured epithelial cells or in vivo, in this study we assayed biofilm formation onto an abiotic surface and compared the results to the ability of our S. maltophilia strains to form biofilm on IB3-1 cells, as assessed by quantitative colony counts. In agreement with previously reported experiments [20, 34], all the twelve S. maltophilia clinical isolates tested were able to form biofilm on both polystyrene and IB3-1 cultured epithelial cells. However, no correlation was found between quantitative biofilm formation on the abiotic surface and qualitative biofilm formation on cultured cell monolayers, thus suggesting that the microtiter plate assay may not be predictive of the ability of S. maltophilia to form biofilm in vivo. Several explanations may account for this discrepancy. The crystal violet assay is surely a less specific method, and it is likely that the dye might also stain negatively charged extracellular molecules, including cell surface molecules and polysaccharides present in the extracellular matrix in mature biofilms, thus influencing the outcome of the test. Further studies are certainly needed to clarify this point.
Recent studies from different laboratories have highlighted the importance of interspecies bacterial interactions in influencing bacterial virulence and response to antibiotic therapy, both in pulmonary infections of CF and non-CF patients [35, 36]. In CF patients, there are several lines of evidence indicating the presence of a mosaic of diverse bacteria so that infections of CF pulmonary tissues are usually considered always polymicrobial . Recently, Ryan et al.  have reported that the presence of S. maltophilia significantly influences, as through the synthesis of a diffusible signal factor, the architecture of P. aeruginosa biofilm formation and augments its susceptibility to polymyxins, recently re-introduced into clinical practice as anti-pseudomonal agents.
In general, S. maltophilia is very often co-isolated with P. aeruginosa from CF patients [6, 25, 39, 40] and it has been hypothesized that infection by P. aeruginosa may enhance the chance of S. maltophilia to colonize CF pulmonary tissues [12, 13]. If this is true, it is reasonable to hypothesize that P. aeruginosa might enhance the ability of S. maltophilia to adhere to and/or invade CF pulmonary tissues. In order to analyze this possibility we first infected IB3-1 cell monolayers with reference P. aeruginosa PAO1 strain, and then with S. maltophilia strain OBGTC9 (the most adhesive of our group of strains; Figure 1A). The results obtained showed that while P. aeruginosa PAO1 binds more efficiently to cell monolayers than does S. maltophilia OBGTC9, a previous exposition of IB3-1 cell monolayers to P. aeruginosa PAO1 significantly improves S. maltophilia adhesiveness; therefore, it suggests a synergistic relationship between these pathogens similarly to what reported by Saiman et al.  who found a synergistic relationship between P. aeruginosa and P. cepacia. Demonstrating this, most (9 out of 12, 75%) of S. maltophilia-positive CF patients considered in the present study was found to have been infected in the past with P. aeruginosa (Table 1).