Gravitropic moss cells default to spiral growth on the clinostat and in microgravity during spaceflight

In addition to shoots and roots, the gravity (g)-vector orients the growth of specialized cells such as the apical cell of dark-grown moss protonemata. Each apical cell of the moss Ceratodon purpureus senses the g-vector and adjusts polar growth accordingly producing entire cultures of upright protonemata (negative gravitropism). The effect of withdrawing a constant gravity stimulus on moss growth was studied on two NASA Space Shuttle (STS) missions as well as during clinostat rotation on earth. Cultures grown in microgravity (spaceflight) on the STS-87 mission exhibited two successive phases of non-random growth and patterning, a radial outgrowth followed by the formation of net clockwise spiral growth. Also, cultures pre-aligned by unilateral light developed clockwise hooks during the subsequent dark period. The second spaceflight experiment flew on STS-107 which disintegrated during its descent on 1 February 2003. However, most of the moss experimental hardware was recovered on the ground, and most cultures, which had been chemically fixed during spaceflight, were retrieved. Almost all intact STS-107 cultures displayed strong spiral growth. Non-random culture growth including clockwise spiral growth was also observed after clinostat rotation. Together these data demonstrate the existence of default non-random growth patterns that develop at a population level in microgravity, a response that must normally be overridden and masked by a constant g-vector on earth.     

Development of the brine shrimp Artemia is accelerated during spaceflight

Developmentally arrested brine shrimp cysts have been reactivated during orbital spaceflight on two different Space Shuttle missions (STS-50 and STS-54), and their subsequent development has been compared with that of simultaneously reactivated ground controls. Flight and control brine shrimp do not significantly differ with respect to hatching rates or larval morphology at the scanning and transmission EM levels. A small percentage of the flight larvae had defective nauplier eye development, but the observation was not statistically significant. However, in three different experiments on two different flights, involving a total of 232 larvae that developed in space, a highly significant difference in degree of flight to control development was found. By as early as 2.25 days after reactivation of development, spaceflight brine shrimp were accelerated, by a full instar, over ground control brine shrimp. Although developing more rapidly, flight shrimp grew as long as control shrimp at each developmental instar or stage.     

Exposure of conjugative plasmid carrying Escherichia coli biofilms to male-specific bacteriophages

Escherichia coli carrying a natural conjugative F-plasmid generates F-pili mating pairs, which is important for early biofilm formation. In this study, we investigated the effect of male-specific filamentous single stranded DNA bacteriophage (f1) and RNA bacteriophage (MS2) on the formation of biofilms by E. coli carrying a natural conjugative F-plasmid. We showed that the early biofilm formation was completely inhibited by addition of the f1 phage, but not the MS2 phage. This suggests that the tip of F-pili is the specific attachment site for mating pairs formation and the side of F-pili has a non-obligatory role during biofilm formation. The inhibitory effect of the f1 phage was dependent on the time of addition during the biofilm formation. No inhibitory effect was observed when the f1 phages were added to the mature biofilms. This resistant mechanism of the mature biofilms could be attributed to the biofilm-specific phenotypes representing that the F-pili mating pairs were already formed and then the curli production commenced during the biofilm maturation. The pre-formed mating pairs seemed to resist the f1 phages. Altogether, our results indicate a close relationship between the presence of conjugative plasmid and male-specific bacteriophages within sessile biofilm communities, as well as the possibility of using the male-specific bacteriophages to control biofilm formation.     

Bacterial biofilm formation under microgravity conditions

Although biofilm formation is widely documented on Earth, it has not been demonstrated in the absence of gravity. To explore this possibility, Pseudomonas aeruginosa, suspended in sterile buffer, was flown in a commercial payload on space shuttle flight STS-95. During earth orbit, biofilm formation was induced by exposing the bacteria to sterile media through a 0.2-microm (pore size) polycarbonate membrane. Examination of these membranes by confocal microscopy revealed biofilms to be present and that these biofilms could persist in spite of vigorous agitation. These results represent the first report of biofilm formation under microgravity conditions.     

Phosphorylcholine expression by nontypeable Haemophilus influenzae correlates with maturation of biofilm communities in vitro and in vivo

Nontypeable Haemophilus influenzae (NTHI) causes chronic infections that feature the formation of biofilm communities. NTHI variants within biofilms have on their surfaces lipooligosaccharides containing sialic acid (NeuAc) and phosphorylcholine (PCho). Our work showed that NeuAc promotes biofilm formation, but we observed no defect in the initial stages of biofilm formation for mutants lacking PCho. In this study, we asked if alterations in NTHI PCho content affect later stages of biofilm maturation. Biofilm communities were compared for NTHI 2019 and isogenic mutants that either lacked PCho (NTHI 2019 licD) or were constitutively locked in the PCho-positive phase (NTHI 2019 lic^ON). Transformants expressing green fluorescent protein were cultured in continuous-flow biofilms and analyzed by confocal laser scanning microscopy. COMSTAT was used to quantify different biofilm parameters. PCho expression correlated significantly with increased biofilm thickness, surface coverage, and total biomass, as well as with a decrease in biofilm roughness. Comparable results were obtained by scanning electron microscopy. Analysis of thin sections of biofilms by transmission electron microscopy revealed shedding of outer membrane vesicles by NTHI bacteria within biofilms and staining of matrix material with ruthenium red in biofilms formed by NTHI 2019 lic^ON. The biofilms of all three strains were comparable in viability, the presence of extracellular DNA, and the presence of sialylated moieties on or between bacteria. In vivo infection studies using the chinchilla model of otitis media showed a direct correlation between PCho expression and biofilm formation within the middle-ear chamber and an inverse relationship between PCho and persistence in the planktonic phase in middle-ear effusions. Collectively, these data show that PCho correlates with, and may promote, the maturation of NTHI biofilms. Further, this structure may be disadvantageous in the planktonic phase.     

Microbial colonization of polymeric materials for space applications and mechanisms of biodeterioration: A review

Biodeterioration of polymeric materials affects a wide range of industries. Formation of microbial biofilms on surfaces of materials being considered for use on the International Space Station was investigated. The materials included fiber-reinforced polymeric composites, adhesive sealant, polyimide insulation foam, Teflon cable insulation, and aliphatic polyurethane coatings. In simulation experiments, bacterial biofilms formed readily on the surfaces of the materials at a wide range of temperatures and relative humidity. The biofilm population was dominated by Pseudomonas aeruginosa, Ochrobactrum anthropi, Alcaligenes denitrificans, Xanthomonas maltophila, and Vibrio harveyi. Subsequently, degradation of polymeric materials was mostly a result of both fungal and bacterial colonization in sequence, and fungi may have advantages in the early phase of surface colonization over bacteria, especially on relatively resistant polymeric materials. These microorganisms are commonly detected on spacecraft on hardware and in the air. Furthermore, degradation of polymeric materials was documented with electrochemical impedance spectroscopy (EIS). The mechanisms of deterioration of polymeric materials were due to the availability of carbon source from the polymer, such as additives, plasticizers, and other impurities, in addition to the polymeric matrices. Microbial degradation of plasticizer phthalate esters is discussed for the microorganisms involved and the biochemical pathways of degradation. Current results suggest that candidate materials for use in space missions need to be carefully evaluated for their susceptibility to microbial biofilm formation and biodegradation.     

High-Throughput Screening of Multispecies Biofilm Formation and Quantitative PCR-Based Assessment of Individual Species Proportions,Useful for Exploring Interspecific Bacterial Interactions

Multispecies biofilms are predominant in almost all natural environments, where myriads of resident microorganisms interact with each other in both synergistic and antagonistic manners. The interspecies interactions among different bacteria are, despite the ubiquity of these communities, still poorly understood. Here, we report a rapid, reproducible and sensitive approach for quantitative screening of biofilm formation by bacteria when cultivated as mono- and multispecies biofilms, based on the Nunc-TSP lid system and crystal violet staining. The relative proportion of the individual species in a four-species biofilm was assessed using quantitative PCR based on SYBR Green I fluorescence with specific primers. The results indicated strong synergistic interactions in a four-species biofilm model community with a more than 3-fold increase in biofilm formation and demonstrated the strong dominance of two strains, Xanthomonas retroflexus and Paenibacillus amylolyticus. The developed approach can be used as a standard procedure for evaluating interspecies interactions in defined microbial communities. This will be of significant value in the quantitative study of the microbial composition of multispecies biofilms both in natural environments and infectious diseases to increase our understanding of the mechanisms that underlie cooperation, competition and fitness of individual species in mixed-species biofilms.     

The biofilm architecture of sixty opportunistic pathogens deciphered using a high throughput CLSM method

This study proposes a high throughput method based on Confocal Laser Scanning Microscopy (CLSM) combined with the use of 96-wells microtiter plates compatible with high resolution imaging for the study of biofilm formation and structure. As an illustration, the three-dimensional structures of biofilms formed by 60 opportunistic pathogens were thus observed and quantified. The results revealed the diversity of biofilm architectures. Specific spatial arrangement such as the mushroom-like structures already described for Pseudomonas aeruginosa was observed. Other features, such as hollow voids in microcolonies of Salmonella enterica strain Agona, were identified for the first time. The combined use of microplates and confocal imaging proved to be a good alternative to the other high throughput methods commonly used as it enables the direct, insitu, qualitative and quantitative characterization of biofilm architecture. This high content method should lead to a clearer understanding of the structure-function relationships implicated in biofilms traits.     

The role of microbial biofilms in deterioration of space station candidate materials

Formation of microbial biofilms on surfaces of a wide range of materials being considered as candidates for use on the International Space Station was investigated. The materials included a fibre-reinforced polymeric composite, an adhesive sealant, a polyimide insulation foam, teflon cable insulation, titanium, and an aliphatic polyurethane coating. They were exposed to a natural mixed population of bacteria under controlled conditions of temperature and relative humidity (RH). Biofilms formed on the surfaces of the materials at a wide range of temperatures and RHs. The biofilm population was dominated by Pseudomonas aeruginosa, Ochrobactrum anthropi, Alcaligenes denitrificans, Xanthomonas maltophila, and Vibrio harveyi. The biocide, diiodomethyl-p-tolyl sulfone, impregnated in the polyurethane coating, was ineffective against microbial colonization and growth. Degradation of the polyurethane coatings was monitored with electrochemical impedance spectroscopy (EIS). The impedance spectra indicated that microbial degradation of the coating occurred in several stages. The initial decreases in impedance were due to the transport of water and solutes into the polymeric matrices. Further decreases were a result of polymer degradation by microorganisms. Our data showed that these candidate materials for space application are susceptible to biofilm formation and subsequent degradation. Our study suggests that candidate materials for use in space missions need to be carefully evaluated for their susceptibility to microbial biofilm formation and biodegradation.     

Enhanced Biofilm Formation and Increased Resistance to Antimicrobial Agents and Bacterial Invasion Are Caused by Synergistic Interactions in Multispecies Biofilms

Most biofilms in their natural environments are likely to consist of consortia of species that influence each other in synergistic and antagonistic manners. However, few reports specifically address interactions within multispecies biofilms. In this study, 17 epiphytic bacterial strains, isolated from the surface of the marine alga Ulva australis, were screened for synergistic interactions within biofilms when present together in different combinations. Four isolates, Microbacterium phyllosphaerae, Shewanella japonica, Dokdonia donghaensis, and Acinetobacter lwoffii, were found to interact synergistically in biofilms formed in 96-well microtiter plates: biofilm biomass was observed to increase by >167% in biofilms formed by the four strains compared to biofilms composed of single strains. When exposed to the antibacterial agent hydrogen peroxide or tetracycline, the relative activity (exposed versus nonexposed biofilms) of the four-species biofilm was markedly higher than that in any of the single-species biofilms. Moreover, in biofilms established on glass surfaces in flow cells and subjected to invasion by the antibacterial protein-producing Pseudoalteromonas tunicata, the four-species biofilms resisted invasion to a greater extent than did the biofilms formed by the single species. Replacement of each strain by its cell-free culture supernatant suggested that synergy was dependent both on species-specific physical interactions between cells and on extracellular secreted factors or less specific interactions. In summary, our data strongly indicate that synergistic effects promote biofilm biomass and resistance of the biofilm to antimicrobial agents and bacterial invasion in multispecies biofilms.     

Experimental and Computational Investigation of Biofilm Formation by Rhodopseudomonas palustris Growth under Two Metabolic Modes

We examined biofilms formed by the metabolically versatile bacterium Rhodopseudomonas palustris grown via different metabolic modes. R. palustris was grown in flow cell chambers with identical medium conditions either in the presence or absence of light and oxygen. In the absence of oxygen and the presence of light, R. palustris grew and formed biofilms photoheterotrophically, and in the presence of oxygen and the absence of light, R. palustris grew and formed biofilms heterotrophically. We used confocal laser scanning microscopy and image analysis software to quantitatively analyze and compare R. palustris biofilm formation over time in these two metabolic modes. We describe quantifiable differences in structure between the biofilms formed by the bacterium grown heterotrophically and those grown photoheterotrophically. We developed a computational model to explore ways in which biotic and abiotic parameters could drive the observed biofilm architectures, as well as a random-forest machine-learning algorithm based on structural differences that was able to identify growth conditions from the confocal imaging of the biofilms with 87% accuracy. Insight into the structure of phototrophic biofilms and conditions that influence biofilm formation is relevant for understanding the generation of biofilm structures with different properties, and for optimizing applications with phototrophic bacteria growing in the biofilm state.     

Quantitative Analysis of Amyloid-Integrated Biofilms Formed by Uropathogenic Escherichia coli at the Air-Liquid Interface

Bacterial biofilms are complex multicellular assemblies, characterized by a heterogeneous extracellular polymeric matrix, that have emerged as hallmarks of persistent infectious diseases. New approaches and quantitative data are needed to elucidate the composition and architecture of biofilms, and such data need to be correlated with mechanical and physicochemical properties that relate to function. We performed a panel of interfacial rheological measurements during biofilm formation at the air-liquid interface by the Escherichia coli strain UTI89, which is noted for its importance in studies of urinary tract infection and for its assembly of functional amyloid fibers termed curli. Brewster-angle microscopy and measurements of the surface elasticity (G_s′) and stress-strain response provided sensitive and quantitative parameters that revealed distinct stages during bacterial colonization, aggregation, and eventual formation of a pellicle at the air-liquid interface. Pellicles that formed under conditions that upregulate curli production exhibited an increase in strength and viscoelastic properties as well as a greater ability to recover from stress-strain perturbation. The results suggest that curli, as hydrophobic extracellular amyloid fibers, enhance the strength, viscoelasticity, and resistance to strain of E. coli biofilms formed at the air-liquid interface.     

Influence of Hydrodynamics and Cell Signaling on the Structure and Behavior of Pseudomonas aeruginosa Biofilms

Biofilms were grown from wild-type (WT) Pseudomonas aeruginosa PAO1 and the cell signaling lasI mutant PAO1-JP1 under laminar and turbulent flows to investigate the relative contributions of hydrodynamics and cell signaling for biofilm formation. Various biofilm morphological parameters were quantified using Image Structure Analyzer software. Multivariate analysis demonstrated that both cell signaling and hydrodynamics significantly (P < 0.000) influenced biofilm structure. In turbulent flow, both biofilms formed streamlined patches, which in some cases developed ripple-like wave structures which flowed downstream along the surface of the flow cell. In laminar flow, both biofilms formed monolayers interspersed with small circular microcolonies. Ripple-like structures also formed in four out of six WT biofilms, although their velocity was approximately 10 times less than that of those that formed in the turbulent flow cells. The movement of biofilm cell clusters over solid surfaces may have important clinical implications for the dissemination of biofilm subject to fluid shear, such as that found in catheters. The ability of the cell signaling mutant to form biofilms in high shear flow demonstrates that signaling mechanisms are not required for the formation of strongly adhered biofilms. Similarity between biofilm morphologies in WT and mutant biofilms suggests that the dilution of signal molecules by mass transfer effects in faster flowing systems mollifies the dramatic influence of signal molecules on biofilm structure reported in previous studies.     

Media Ion Composition Controls Regulatory and Virulence Response of Salmonella in Spaceflight

The spaceflight environment is relevant to conditions encountered by pathogens during the course of infection and induces novel changes in microbial pathogenesis not observed using conventional methods. It is unclear how microbial cells sense spaceflight-associated changes to their growth environment and orchestrate corresponding changes in molecular and physiological phenotypes relevant to the infection process. Here we report that spaceflight-induced increases in Salmonella virulence are regulated by media ion composition, and that phosphate ion is sufficient to alter related pathogenesis responses in a spaceflight analogue model. Using whole genome microarray and proteomic analyses from two independent Space Shuttle missions, we identified evolutionarily conserved molecular pathways in Salmonella that respond to spaceflight under all media compositions tested. Identification of conserved regulatory paradigms opens new avenues to control microbial responses during the infection process and holds promise to provide an improved understanding of human health and disease on Earth.     

Biofilm formation on human airway epithelia by encapsulated Neisseria meningitidis serogroup B

Neisseria meningitidis is the etiologic agent of meningococcal meningitis. We compared 48-h biofilm formation by N. meningitidis serogroup B strains NMB, MC58, C311 and isogenic mutants defective in capsule formation on SV-40 transformed human bronchial epithelial (HBE) cells in a flow cell. We demonstrated that strains NMB and NMB siaA-D were defective in biofilm formation over glass, and there was a partial rescue of biofilm growth for strain NMB on collagen-coated coverslips at 48 h. We demonstrated all three serogroup B strains form biofilms of statistically equivalent average height on HBE cells as their isogenic capsular mutants. Strain NMB also formed a biofilm of statistically equivalent biomass as the NMB siaA-D mutant on HBE cells at 6 and 48 h. These biofilms are significantly larger than biofilms formed over glass or collagen. Verification that strain NMB expressed capsule in biofilms on HBE cells was demonstrated by staining with 2.2.B, a monoclonal antibody with specificity for the serogroup B capsule. ELISA analysis demonstrated that strains MC58 and C311 also produced capsules during biofilm growth. These findings suggest that encapsulated meningococci can form biofilms on epithelial cells suggesting that biofilm formation may play a role in nasopharyngeal colonization.     

PslG,a self-produced glycosyl hydrolase,triggers biofilm disassembly by disrupting exopolysaccharide matrix

Biofilms are surface-associated communities of microorganism embedded in extracellular matrix. Exopolysaccharide is a critical component in the extracellular matrix that maintains biofilm architecture and protects resident biofilm bacteria from antimicrobials and host immune attack. However, self-produced factors that target the matrix exopolysaccharides, are still poorly understood. Here, we show that PslG, a protein involved in the synthesis of a key biofilm matrix exopolysaccharide Psl in Pseudomonas aeruginosa, prevents biofilm formation and disassembles existing biofilms within minutes at nanomolar concentrations when supplied exogenously. The crystal structure of PslG indicates the typical features of an endoglycosidase. PslG mainly disrupts the Psl matrix to disperse bacteria from biofilms. PslG treatment markedly enhances biofilm sensitivity to antibiotics and macrophage cells, resulting in improved biofilm clearance in a mouse implant infection model. Furthermore, PslG shows biofilm inhibition and disassembly activity against a wide range of Pseudomonas species, indicating its great potential in combating biofilm-related complications.     

NASDA aquatic animal experiment facilities for Space Shuttle.

National Space Development Agency of Japan (NASDA) has been developed aquatic animal experiment facilities for space experiments using NASA Space Shuttle. Vestibular Function Experiment Unit (VFEU) has been firstly designed and developed for Spacelab-J mission (STS-47), and 8 days space experiment with carp has been performed. Following, the VFEU, Aquatic Animal Experiment Unit (AAEU) has been developed to accommodate small aquatic animals second International Microgravity Laboratory mission (IML-2, STS-65). Four kinds of space experiments with goldfish, medaka, newt, and newt eggs have been performed for 15 days mission duration. Then, VFEU has been improved to accommodate marine fish under low temperature condition for Neurolab (STS-90) and STS-95 missions. 17 days (STS-90) and 9 days (STS-95) experiments with oyster toadfish have been performed by using the VFEU. This report summarizes the outline of these aquatic animal experiment facilities.     

Extracellular DNA Inhibits Salmonella enterica Serovar Typhimurium and S. enterica Serovar Typhi Biofilm Development on Abiotic Surfaces

Extracellular DNA (eDNA) was identified and characterized in a 2-day-old biofilms developed by Salmonella enterica ser. Typhimurium SR-11 and S. enterica ser. Typhi ST6 using confocal laser scanning microscopy (CLSM) and enzymatic extraction methods. Results of microtitre plate assay and CLSM analysis showed both Salmonella strains formed significantly more biofilms in the presence of DNase I; Furthermore, a remarkable decrease of biofilm formation was observed when eDNA was added in the inoculation. However, for the pre-established biofilms on polystyrene and glass, no significant difference was observed between the DNase I treated biofilm and the corresponding non-treated controls. In conclusion, these results demonstrate that eDNA is a novel matrix component of Salmonella biofilms. This is the first evidence for the presence of eDNA and its inhibitive and destabilizing effect during biofilm development of S. enterica ser. Typhimurium and S. enterica ser. Typhi on abiotic surfaces.     

Role of Mutation in Pseudomonas aeruginosa Biofilm Development

The survival of bacteria in nature is greatly enhanced by their ability to grow within surface-associated communities called biofilms. Commonly, biofilms generate proliferations of bacterial cells, called microcolonies, which are highly recalcitrant, 3-dimensional foci of bacterial growth. Microcolony growth is initiated by only a subpopulation of bacteria within biofilms, but processes responsible for this differentiation remain poorly understood. Under conditions of crowding and intense competition between bacteria within biofilms, microevolutionary processes such as mutation selection may be important for growth; however their influence on microcolony-based biofilm growth and architecture have not previously been explored. To study mutation in-situ within biofilms, we transformed Pseudomonas aeruginosa cells with a green fluorescent protein gene containing a +1 frameshift mutation. Transformed P. aeruginosa cells were non-fluorescent until a mutation causing reversion to the wildtype sequence occurs. Fluorescence-inducing mutations were observed in microcolony structures, but not in other biofilm cells, or in planktonic cultures of P. aeruginosa cells. Thus microcolonies may represent important foci for mutation and evolution within biofilms. We calculated that microcolony-specific increases in mutation frequency were at least 100-fold compared with planktonically grown cultures. We also observed that mutator phenotypes can enhance microcolony-based growth of P. aeruginosa cells. For P. aeruginosa strains defective in DNA fidelity and error repair, we found that microcolony initiation and growth was enhanced with increased mutation frequency of the organism. We suggest that microcolony-based growth can involve mutation and subsequent selection of mutants better adapted to grow on surfaces within crowded-cell environments. This model for biofilm growth is analogous to mutation selection that occurs during neoplastic progression and tumor development, and may help to explain why structural and genetic heterogeneity are characteristic features of bacterial biofilm populations.