Biofilm formation by Legionella spp. in experiment

Ability of biofilm formation was studied in 28 strains belonging to 12 species of Legionella. Optimal conditions for formation of biofilms were ascertained using reference strain Legionella pneumophila Philadelphia 1. Comparative assessment of the ability of Legionella spp. to form biofilms was performed by cultivation in proteosopepton broth (for 96 hours) and in water (for up to 2 weeks). Highest rates of biofilm formation were observed for strains of L. pneumophila and L. longbeachae. Between L. pneumophila strains the most prominent ability to form biofilms was observed in newly isolated strains BLR-05 and TOTAL 1. Opportunity to use different ability of Legionella species to biofilm formation as a epidemiologically significant marker and for modeling of biofilms of Legionella in association with other microorganisms was discussed.     

Interspecies biofilms of Pseudomonas aeruginosa and Burkholderia cepacia.

The leading cause of morbidity and mortality in cystic fibrosis (CF) continues to be lung infections with Pseudomonas aeruginosa biofilms. Co-colonization of the lungs with P aeruginosa and Burkholderia cepacia can result in more severe pulmonary disease than P. aeruginosa alone. The interactions between P. aeruginosa biofilms and B. cepacia are not yet understood; one possible association being that mixed species biofilm formation may be part of the interspecies relationship. Using the Calgary Biofilm Device (CBD), members of all genomovars of the B. cepacia complex were shown to form biofilms, including those isolated from CF lungs. Mixed species biofilm formation between CF isolates of P. aeruginosa and B. cepacia was readily achieved using the CBD. Oxidation-fermentation lactose agar was adapted as a differential agar to monitor mixed biofilm composition. Scanning electron micrographs of the biofilms demonstrated that both species readily integrated in close association in the biofilm structure. Pseudomonas aeruginosa laboratory strain PAO1, however, inhibited mixed biofilm formation of both CF isolates and environmental strains of the B. cepacia complex. Characterization of the soluble inhibitor suggested pyocyanin as the active compound.     

Assessment of bactericidal activity of disinfectants against Legionella on the biofilm model

Comparative assessment of bactericidal activity of different disinfectants against Legionella biofilms was conducted. Monospecies biofilms of 3 strains of Legionella pneumophila obtained on plastic plates in stable conditions were used as models. It has been shown that for degradation of biofilms as well as for prophylactic action of disinfectants in preventing formation of biofilms on plastic surfaces, higher concentrations of preparations were needed as compared to their bactericidal concentrations for culture of Legionella determined by method of serial dilutions.     

Association patterns of Pseudomonas aeruginosa clinical isolates as revealed by virulence traits, antibiotic resistance, serotype and genotype

The aims of this study were to assess the association patterns of 96 clinical isolates of Pseudomonas aeruginosa using hierarchical cluster analysis from data obtained from the measurement of the physicochemical cell surface properties, adhesion and initial biofilm formation abilities, to investigate any correspondence with source, serotype, beta-lactam pattern, motility and M13-PCR genogroup or clonal lineage, as well as to select clinical isolates that could act as representatives of the genotypic and phenotypic diversity of this P. aeruginosa population from a Portuguese Central Hospital. The isolates were phenotypically characterized by their ability to adhere and form biofilms on polystyrene surfaces, their affinity to hexadecane and silicone, their swimming and twitching abilities, their antibiotic susceptibility patterns and their serotypes. No particular phenotypic cluster associated with the same source, serotype, beta-lactam pattern, motility and M13-PCR genogroup and clonal lineage was found. Nevertheless, five representative strains of the P. aeruginosa population from this Hospital, selected on the basis of low genetic similarity, were also found to be dispersed among the phenotypic clusters.     

Sessile Legionella pneumophila is able to grow on surfaces and generate structured monospecies biofilms

Currently, models for studying Legionella pneumophila biofilm formation rely on multi-species biofilms with low reproducibility or on growth in rich medium, where planktonic growth is unavoidable. The present study describes a new medium adapted to the growth of L. pneumophila monospecies biofilms in vitro. A microplate model was used to test several media. After incubation for 6 days in a specific biofilm broth not supporting planktonic growth, biofilms consisted of 5.36 ± 0.40 log (cfu cm^?2) or 5.34 ± 0.33 log (gu cm^?2). The adhered population remained stable for up to 3 weeks after initial inoculation. In situ confocal microscope observations revealed a typical biofilm structure, comprising cell clusters ranging up to ～300 μm in height. This model is adapted to growing monospecies L. pneumophila biofilms that are structurally different from biofilms formed in a rich medium. High reproducibility and the absence of other microbial species make this model useful for studying genes involved in biofilm formation.     

Establishment and Early Succession of a Multispecies Biofilm Composed of Soil Bacteria

Most soil bacteria are likely to be organized in biofilms on roots, litter, or soil particles. Studies of such biofilms are complicated by the many nonculturable species present in soil, as well as the interspecific bacterial interactions affecting biofilm biology. We in this study describe the development of a biofilm flow model and use this system to establish an early (days 1–7) flow biofilm of soil bacteria from agricultural soil. It was possible to follow the succession in the early flow biofilm by denaturing gradient gel electrophoresis (DGGE) analysis, and it was demonstrated that the majority of strains present in the biofilm were culturable. We isolated and identified nine strains, all associated with unique DGGE profiles, and related their intrinsic phenotypes regarding monospecies biofilm formation in microtiter plates and planktonic growth characteristics to the appearance of the strains in the flow biofilm. The ability of the strains to attach to and establish biofilm in microtiter plates was reflected in their flow biofilm appearance, whereas no such reflection of the planktonic growth characteristics in the flow biofilm appearance was observed. One strain-specific synergistic interaction, strongly promoting biofilm formation of two strains when cultured together in a dual-species biofilm, was observed, indicating that some strains promote biofilm formation of others. Thus, the biofilm flow model proved useful for investigations of how intrinsic phenotypic traits of individual species affect the succession in an early soil biofilm consortium.     

Legionella: from environmental habitats to disease pathology, detection and control

Studies on Legionella show a continuum from environment to human disease. Legionellosis is caused by Legionella species acquired from environmental sources, principally water sources such as cooling towers, where Legionella grows intracellularly in protozoa within biofilms. Aquatic biofilms, which are widespread not only in nature, but also in medical and dental devices, are ecological niches in which Legionella survives and proliferates and the ultimate sources to which outbreaks of legionellosis can be traced. Invasion and intracellular replication of L. pneumophila within protozoa in the environment play a major role in the transmission of Legionnaires' disease. Protozoa provide the habitats for the environmental survival and reproduction of Legionella species. L. pneumophila proliferates intracellularly in various species of protozoa within vacuoles studded with ribosomes, as it also does within macrophages. Growth within protozoa enhances the environmental survival capability and the pathogenicity (virulence) of Legionella . The growth requirements of Legionella , the ability of Legionella to enter a viable non-culturable state, the association of Legionella with protozoa and the occurrence of Legionella within biofilms complicates the detection of Legionella and epidemiological investigations of legionellosis. Polymerase chain reaction (PCR) methods have been developed for the molecular detection of Legionella and used in environmental and epidemiological studies. Various physical and chemical disinfection methods have been developed to eliminate Legionella from environmental sources, but gaining control of Legionella in environmental waters, where they are protected from disinfection by growing within protozoa and biofilms, remains a challenge, and one that must be overcome in order to eliminate sporadic outbreaks of legionellosis.     

Immigration and emigration of Burkholderia cepacia and Pseudomonas aeruginosa between and within mixed biofilm communities

AIMS: To investigate the dynamics of binary culture biofilm formation through use of both the Sorbarod model of biofilm growth and the constant depth film fermenter (CDFF). METHODS AND RESULTS: Pseudo steady-state biofilm cultures of laboratory and clinical strains of Pseudomonas aeruginosa, selected on the basis of their ability to produce a Burkholderia cepacia growth-inhibitory substance, were established on Sorbarod filters and challenged with corresponding planktonic grown cultures of B. cepacia. Reverse challenges were also conducted. Both B. cepacia and P. aeruginosa were able to form steady-state monoculture biofilms after 48 h growth. When steady-state biofilms of B. cepacia NTCT 10661 were challenged with planktonically grown P. aeruginosa PAO1 known to produce a B. cepacia growth-inhibitory substance, the immigrant population was rapidly and almost completely bound to the biofilm, displacing B. cepacia. By contrast, established biofilms of P. aeruginosa PAO1 resisted immigration of B. cepacia 10661. Similar experiments conducted with a nongrowth inhibitory substance producing clinical pairing of P. aeruginosa 313113 and B. cepacia 313113 led to the formation of stable, mixed biofilm populations in both instances. Moreover, co-inoculation with these clinical isolates resulted in a stable, mixed steady-state biofilm. Similar observations were made for biofilms generated in CDFFs. In such instances following pan-swapping between two monoculture CDFFs, B. cepacia 313113 was able to integrate into an established P. aeruginosa 313113 biofilm to form a stable binary biofilm. CONCLUSIONS: Establishment of a mixed species community follows a specific sequence of inoculation that may either be due to some degree of match between co-colonizers or that P. aeruginosa predisposes uncolonized sections of the surface to permit B. cepacia colonization. SIGNIFICANCE AND IMPACT OF THE STUDY: Colonization of a surface with one bacterial species confers colonization resistance towards other species. Disinfection of a surface might well increase the probability of pathogen harbourage.     

Structure and species composition of mercury-reducing biofilms

Mercury-reducing biofilms from packed-bed bioreactors treating nonsterile industrial effluents were shown to consist of a monolayer of bacteria by scanning electron microscopy. Droplets of several micrometers in diameter which accumulated outside of the bacterial cells were identified as elemental mercury by electron-dispersive X-ray analysis. The monospecies biofilms of Pseudomonas putida Spi3 initially present were invaded by additional strains, which were identified to the species level by thermogradient gel electrophoresis (TGGE) and 16S rDNA sequencing. TGGE community fingerprints of the biofilms showed that they were composed of the effluent bacteria and did not contain uncultivable microorganisms. Of the 13 effluent bacterial strains, 2 were not mercury resistant, while all the others had resistance levels similar to or higher than the inoculant strain.     

Biofilms, infectious agents, and dental unit waterlines: a review

Aquatic biofilms, which are widespread not only in nature but also in medical and dental devices, can be the source of serious nosocomial infections. In these hardy microbial communities, pathogens like nontuberculous mycobacteria, Pseudomonas aeruginosa, Legionella pneumophila, and other bacteria not only survive but proliferate and lie in wait for susceptible hosts. Not only are these organisms intrinsically resistant to high temperatures and biocides, but the biofilms they inhabit enhance their resistance. This should be of concern to infection control practitioners. The bacterial colonization of dental unit waterlines can be used as a model to investigate the problem of waterborne biofilms in health care settings.     

Characterization of biofilm formation by clinically relevant serotypes of group A streptococci

Streptococcus pyogenes (group A streptococcus [GAS]) is a frequent cause of purulent infections in humans. As potentially important aspects of its pathogenicity, GAS was recently shown to aggregate, form intratissue microcolonies, and potentially participate in multispecies biofilms. In this study, we show that GAS in fact forms monospecies biofilms in vitro, and we analyze the basic parameters of S. pyogenes in vitro biofilm formation, using Streptococcus epidermidis as a biofilm-positive control. Of nine clinically important serotype strains, M2, M6, M14, and M18 were found to significantly adhere to coated and uncoated polystyrene surfaces. Fibronectin and collagen types I and IV best supported primary adherence of serotype M2 and M18 strains, respectively, whereas serotype M6 and M14 strains strongly bound to uncoated polystyrene surfaces. Absorption measurements of safranin staining, as well as electron scanning and confocal laser scanning microscopy, documented that primary adherence led to subsequent formation of three-dimensional biofilm structures consisting of up to 46 bacterial layers. Of note, GAS isolates belonging to the same serotype were found to be very heterogeneous in their biofilm-forming behavior. Biofilm formation was equally efficient under static and continuous flow conditions and consisted of the classical three steps, including partial disintegration after long-term incubation. Activity of the SilC signaling peptide as a component of a putative quorum-sensing system was found to influence the biofilm structure and density of serotype M14 and M18 strains. Based on the presented methods and results, standardized analyses of GAS biofilms and their impact on GAS pathogenicity are now feasible.     

Rapid evaluation of the antibiotic susceptibility of fuel ethanol contaminant biofilms

Bacterial contaminants from commercial fuel ethanol production facilities were previously shown to form biofilms as mixed cultures under laboratory conditions. In this study, a rapid assay was developed to simultaneously compare isolates for their ability to form biofilms as pure cultures. A total of 10 strains were isolated from a dry-grind fuel ethanol plant that routinely doses with virginiamycin. These were identified by sequence analysis as six strains of Lactobacillus fermentum, two strains of L. johnsonii, and one strain each of L. mucosae and L. amylovorus. Isolates exhibited a range of susceptibility to virginiamycin in a planktonic assay, with MIC’s (minimum inhibitory concentration) of ?0.5-16 μg/ml. Even though all strains were isolated from a mixed culture biofilm, they varied greatly in their ability to form biofilms as pure cultures. Surprisingly, growth as biofilms did not appear to provide resistance to virginiamycin, even if biofilms were grown for 144 h prior to antibiotic challenge.     

Adsorption, attachment and biofilm formation among isolates of Listeria monocytogenes using model conditions

Aims:  To determine whether isolates of Listeria monocytogenes differ in their ability to adsorb and form biofilms on a food-grade stainless steel surface.
Methods and Results:  Strains were assessed for their ability to adsorb to a test surface over a short time period. Although some differences in numbers of bound cells were found among the strains, there were no correlations between the degree of adsorption and either the serotype or source of the strain. The ability of each strain to form a biofilm when grown with the test surface was also assessed. With the exception of a single strain, all strains adhered as single cells and did not form biofilms. Significant differences in adherence levels were found among strains. Strains demonstrating enhanced attachment produced extracellular fibrils, whereas those which adhered poorly did not. A single strain formed a biofilm consisting of adhered single cells and aggregates of cells.
Conclusions:  Significant differences were found in the ability of various L. monocytogenes strains to attach to a test surface. In monoculture, the majority of strains did not form biofilms.
Significance and Impact of the Study:  Differences in attachment and biofilm formation among strains provide a basis to study these characteristics in L. monocytogenes .     

The exopolysaccharide alginate protects Pseudomonas aeruginosa biofilm bacteria from IFN-gamma-mediated macrophage killing

The ability of Pseudomonas aeruginosa to form biofilms and cause chronic infections in the lungs of cystic fibrosis patients is well documented. Numerous studies have revealed that P. aeruginosa biofilms are highly refractory to antibiotics. However, dramatically fewer studies have addressed P. aeruginosa biofilm resistance to the host's immune system. In planktonic, unattached (nonbiofilm) P. aeruginosa, the exopolysaccharide alginate provides protection against a variety of host factors yet the role of alginate in protection of biofilm bacteria is unclear. To address this issue, we tested wild-type strains PAO1, PA14, the mucoid cystic fibrosis isolate, FRD1 (mucA22+), and the respective isogenic mutants which lacked the ability to produce alginate, for their susceptibility to human leukocytes in the presence and absence of IFN-gamma. Human leukocytes, in the presence of recombinant human IFN-gamma, killed biofilm bacteria lacking alginate after a 4-h challenge at 37 degrees C. Bacterial killing was dependent on the presence of IFN-gamma. Killing of the alginate-negative biofilm bacteria was mediated through mononuclear cell phagocytosis since treatment with cytochalasin B, which prevents actin polymerization, inhibited leukocyte-specific bacterial killing. By direct microscopic observation, phagocytosis of alginate-negative biofilm bacteria was significantly increased in the presence of IFN-gamma vs all other treatments. Addition of exogenous, purified alginate to the alginate-negative biofilms restored resistance to human leukocyte killing. Our results suggest that although alginate may not play a significant role in bacterial attachment, biofilm development, and formation, it may play an important role in protecting mucoid P. aeruginosa biofilm bacteria from the human immune system.     

Structure and Species Composition of Mercury-Reducing Biofilms

Mercury-reducing biofilms from packed-bed bioreactors treating nonsterile industrial effluents were shown to consist of a monolayer of bacteria by scanning electron microscopy. Droplets of several micrometers in diameter which accumulated outside of the bacterial cells were identified as elemental mercury by electron-dispersive X-ray analysis. The monospecies biofilms of Pseudomonas putida Spi3 initially present were invaded by additional strains, which were identified to the species level by thermogradient gel electrophoresis (TGGE) and 16S rDNA sequencing. TGGE community fingerprints of the biofilms showed that they were composed of the effluent bacteria and did not contain uncultivable microorganisms. Of the 13 effluent bacterial strains, 2 were not mercury resistant, while all the others had resistance levels similar to or higher than the inoculant strain.     

Multiple roles of biosurfactants in structural biofilm development by Pseudomonas aeruginosa

Recent studies have indicated that biosurfactants produced by Pseudomonas aeruginosa play a role both in maintaining channels between multicellular structures in biofilms and in dispersal of cells from biofilms. Through the use of flow cell technology and enhanced confocal laser scanning microscopy, we have obtained results which suggest that the biosurfactants produced by P. aeruginosa play additional roles in structural biofilm development. We present genetic evidence that during biofilm development by P. aeruginosa, biosurfactants promote microcolony formation in the initial phase and facilitate migration-dependent structural development in the later phase. P. aeruginosa rhlA mutants, deficient in synthesis of biosurfactants, were not capable of forming microcolonies in the initial phase of biofilm formation. Experiments involving two-color-coded mixed-strain biofilms showed that P. aeruginosa rhlA mutants were defective in migration-dependent development of mushroom-shaped multicellular structures in the later phase of biofilm formation. Experiments involving three-color-coded mixed-strain P. aeruginosa biofilms demonstrated that the wild-type and rhlA and pilA mutant strains formed distinct subpopulations on top of each other dependent on their ability to migrate and produce biosurfactants.     

Role of exopolysaccharides in Pseudomonas aeruginosa biofilm formation and architecture

Pseudomonas aeruginosa is an opportunistic human pathogen and has been established as a model organism to study bacterial biofilm formation. At least three exopolysaccharides (alginate, Psl, and Pel) contribute to the formation of biofilms in this organism. Here mutants deficient in the production of one or more of these polysaccharides were generated to investigate how these polymers interactively contribute to biofilm formation. Confocal laser scanning microscopy of biofilms formed in flow chambers showed that mutants deficient in alginate biosynthesis developed biofilms with a decreased proportion of viable cells than alginate-producing strains, indicating a role of alginate in viability of cells in biofilms. Alginate-deficient mutants showed enhanced extracellular DNA (eDNA)-containing surface structures impacting the biofilm architecture. PAO1 ΔpslA Δalg8 overproduced Pel, and eDNA showing meshwork-like structures presumably based on an interaction between both polymers were observed. The formation of characteristic mushroom-like structures required both Psl and alginate, whereas Pel appeared to play a role in biofilm cell density and/or the compactness of the biofilm. Mutants producing only alginate, i.e., mutants deficient in both Psl and Pel production, lost their ability to form biofilms. A lack of Psl enhanced the production of Pel, and the absence of Pel enhanced the production of alginate. The function of Psl in attachment was independent of alginate and Pel. A 30% decrease in Psl promoter activity in the alginate-overproducing MucA-negative mutant PDO300 suggested inverse regulation of both biosynthesis operons. Overall, this study demonstrated that the various exopolysaccharides and eDNA interactively contribute to the biofilm architecture of P. aeruginosa.     

The roles of biofilm matrix polysaccharide Psl in mucoid Pseudomonas aeruginosa biofilms

The opportunistic pathogen Pseudomonas aeruginosa causes life-threatening, persistent infections in patients with cystic fibrosis (CF). Persistence is attributed to the ability of these bacteria to form structured communities (biofilms). Biofilms rely on an extracellular polymeric substances matrix to maintain structure. Psl exopolysaccharide is a key matrix component of nonmucoid biofilms, yet the role of Psl in mucoid biofilms is unknown. In this report, using a variety of mutants in a mucoid P.?aeruginosa background, we found that deletion of Psl-encoding genes dramatically decreased their biofilm formation ability, indicating that Psl is also a critical matrix component of mucoid biofilms. Our data also suggest that the overproduction of alginate leads to mucoid biofilms, which occupy more space, whereas Psl-dependent biofilms are densely packed. These data suggest that Psl polysaccharide may have significant contributions in biofilm persistence in patients with CF and may be helpful for designing therapies for P.?aeruginosa CF infection.     

Temperature-regulated formation of mycelial mat-like biofilms by Legionella pneumophila

Fifty strains representing 38 species of the genus Legionella were examined for biofilm formation on glass, polystyrene, and polypropylene surfaces in static cultures at 25 degrees C, 37 degrees C, and 42 degrees C. Strains of Legionella pneumophila, the most common causative agent of Legionnaires' disease, were found to have the highest ability to form biofilms among the test strains. The quantity, rate of formation, and adherence stability of L. pneumophila biofilms showed considerable dependence on both temperature and surface material. Glass and polystyrene surfaces gave between two- to sevenfold-higher yields of biofilms at 37 degrees C or 42 degrees C than at 25 degrees C; conversely, polypropylene surface had between 2 to 16 times higher yields at 25 degrees C than at 37 degrees C or 42 degrees C. On glass surfaces, the biofilms were formed faster but attached less stably at 37 degrees C or 42 degrees C than at 25 degrees C. Both scanning electron microscopy and confocal laser scanning microscopy revealed that biofilms formed at 37 degrees C or 42 degrees C were mycelial mat like and were composed of filamentous cells, while at 25 degrees C, cells were rod shaped. Planktonic cells outside of biofilms or in shaken liquid cultures were rod shaped. Notably, the filamentous cells were found to be multinucleate and lacking septa, but a recA null mutant of L. pneumophila was unaffected in its temperature-regulated filamentation within biofilms. Our data also showed that filamentous cells were able to rapidly give rise to a large number of short rods in a fresh liquid culture at 37 degrees C. The possibility of this biofilm to represent a novel strategy by L. pneumophila to compete for proliferation among the environmental microbiota is discussed.     

Biofilm-growing intestinal anaerobic bacteria

Sessile growth of anaerobic bacteria from the human intestinal tract has been poorly investigated, so far. We recently reported data on the close association existing between biliary stent clogging and polymicrobial biofilm development in its lumen. By exploiting the explanted stents as a rich source of anaerobic bacterial strains belonging to the genera Bacteroides, Clostridium, Fusobacterium, Finegoldia, Prevotella, and Veillonella, the present study focused on their ability to adhere, to grow in sessile mode and to form in vitro mono- or dual-species biofilms. Experiments on dual-species biofilm formation were planned on the basis of the anaerobic strains isolated from each clogged biliary stent, by selecting those in which a couple of anaerobic strains belonging to different species contributed to the polymicrobial biofilm development. Then, strains were investigated by field emission scanning electron microscopy and confocal laser scanning microscopy to reveal if they are able to grow as mono- and/or dual-species biofilms. As far as we know, this is the first report on the ability to adhere and form mono/dual-species biofilms exhibited by strains belonging to the species Bacteroides oralis, Clostridium difficile, Clostridium baratii, Clostridium fallax, Clostridium bifermentans, Finegoldia magna, and Fusobacterium necrophorum.