Biofilm Formation on Reverse Osmosis Membranes Is Initiated and Dominated by Sphingomonas spp.

The initial formation and spatiotemporal development of microbial biofilm layers on surfaces of new and clean reverse osmosis (RO) membranes and feed-side spacers were monitored in situ using flow cells placed in parallel with the RO system of a full-scale water treatment plant. The feed water of the RO system had been treated by the sequential application of coagulation, flocculation, sand filtration, ultrafiltration, and cartridge filtration processes. The design of the flow cells permitted the production of permeate under cross-flow conditions similar to those in spiral-wound RO membrane elements of the full-scale system. Membrane autopsies were done after 4, 8, 16, and 32 days of flow-cell operation. A combination of molecular (fluorescence in situ hybridization [FISH], denaturing gradient gel electrophoresis [DGGE], and cloning) and microscopic (field emission scanning electron, epifluorescence, and confocal laser scanning microscopy) techniques was applied to analyze the abundance, composition, architecture, and three-dimensional structure of biofilm communities. The results of the study point out the unique role of Sphingomonas spp. in the initial formation and subsequent maturation of biofilms on the RO membrane and feed-side spacer surfaces.In the water production industry, reverse osmosis (RO) membrane technology is a durable, promising, and much-used separation method. Its application enables the efficient removal of a wide variety of contaminants (i.e., microbial constituents, total dissolved solids, and organic compounds). Feed streams of different qualities (e.g., raw, natural, chemically contaminated or brackish, and seawater) are used to produce high-purity water that is microbiologically safe and biologically stable (15, 25). However, the widespread application of this technology is limited because the current generation of RO filtration units experience biofouling problems (14). The design of so-called “spiral wound” membrane elements and the conditions at the membrane, feed-side spacer, and other internal surfaces within these RO filters make them prone to microbial attachment and the subsequent formation of biofilm layers. A variety of microorganisms are involved in the development of these surface-attached complex structures after prolonged operation of the RO system, depending on the type and concentration of contaminants in the feed water and the type of pretreatment (5, 6, 7, 32, 38). The biofilm occurrence is a principal problem for proper RO system performance. It can lead to blocking of the feed concentrate channel and to clogging of the membrane. Biofilm formation results in an increased energy requirement of the feed water pumps, a lower flux, and a decrease of permeate quality (14). Conventional prevention and/or management strategies of biofouling-caused problems require more frequent chemical cleanings, thereby leading to a shortened membrane life and, ultimately, to a loss of capacity of the water supply plant (3, 14). Finding more effective ways to deal with biofouling problems in the current RO systems still needs more fundamental investigations of all aspects of biofilm formation. Little is known about the microbial community that makes up the biofilm on the membranes. To diagnose biofouling and to choose the most appropriate pretreatment and cleaning strategies, the pressure difference between the inlet and outlet channels and microbial biomass concentrations can be determined (48). Additional microbiological research, such as total cell and heterotrophic plate counts, provides some basic information (12, 23). However, such experiments do not allow for a reliable evaluation of microbial abundance and diversity of species, because the majority of the microorganisms in ecosystems cannot be cultured (21). While knowledge of real biofilm microbial composition is essential in identifying the most effective cleaning protocols, only a few molecular-based microbial diversity studies on RO membrane surfaces are reported (5, 6, 7, 32). In addition, limited data about the formation and development of biofilms over time are available. What little is known comes from laboratory-controlled biofilm monitoring studies using one or a few bacterial strains for biofilm formation (18, 19). These studies, therefore, may not provide a true representation of the RO biofilm problem in situ.In this study, we investigated microbial biofilm formation in an experimental setup similar to an authentic RO system. Using stainless steel flow cells connected in parallel to the reverse osmosis system of a full-scale water treatment plant, the spatiotemporal development of microbial biofilms on the surfaces of new and clean reverse osmosis membranes and feed-side spacers was monitored. The bacteria responsible for the initial colonization and development of the biofilms were identified by various molecular and microscopic techniques.     

Molecular characterization of the bacterial communities in the different compartments of a full-scale reverse-osmosis water purification plant

The origin, structure, and composition of biofilms in various compartments of an industrial full-scale reverse-osmosis (RO) membrane water purification plant were analyzed by molecular biological methods. Samples were taken when the RO installation suffered from a substantial pressure drop and decreased production. The bacterial community of the RO membrane biofilm was clearly different from the bacterial community present at other locations in the RO plant, indicating the development of a specialized bacterial community on the RO membranes. The typical freshwater phylotypes in the RO membrane biofilm (i.e., Proteobacteria, Cytophaga-Flexibacter-Bacteroides group, and Firmicutes) were also present in the water sample fed to the plant, suggesting a feed water origin. However, the relative abundances of the different species in the mature biofilm were different from those in the feed water, indicating that the biofilm was actively formed on the RO membrane sheets and was not the result of a concentration of bacteria present in the feed water. The majority of the microorganisms (59% of the total number of clones) in the biofilm were related to the class Proteobacteria, with a dominance of Sphingomonas spp. (27% of all clones). Members of the genus Sphingomonas seem to be responsible for the biofouling of the membranes in the RO installation.     

Effects of seawater ozonation on biofilm development in aquaculture tanks

Microbial biofilms developing in aquaculture tanks represent a reservoir for opportunistic bacterial pathogens, and procedures to control formation and bacterial composition of biofilms are important for the development of commercially viable aquaculture industries. This study investigated the effects of seawater ozonation on biofilm development on microscope glass slides placed in small-scale aquaculture tanks containing the live feed organism Artemia. Fluorescence in situ hybridization (FISH) demonstrated that ozonation accelerated the biofilm formation cycle, while it delayed the establishment of filamentous bacteria. Gammaproteobacteria and Alphaproteobacteria were the most abundant bacterial groups in the biofilm for both water types, but ozonation influenced their dynamics. With ozonation, the bacterial community structure was relatively stable and dominated by Gammaproteobacteria throughout the experiment (21–66% of total bacteria). Without ozonation, the community showed larger fluctuations, and Alphaproteobacteria emerged as dominant after 18 days (up to 54% of total bacteria). Ozonation of seawater also affected the dynamics of less abundant populations in the biofilm such as Betaproteobacteria, Planctomycetales and the Cytophaga/Flavobacterium branch of phylum Bacteroidetes. The abundance of Thiothrix, a bacterial genus capable of filamentous growth and fouling of larvae, increased with time for both water types, while no temporal trend could be detected for the genus Vibrio. Denaturing gradient gel electrophoresis (DGGE) demonstrated temporal changes in the dominant bacterial populations for both water types. Sequencing of DGGE bands confirmed the FISH data, and sequences were related to bacterial groups commonly found in biofilms of aquaculture systems. Several populations were closely related to organisms involved in sulfur cycling. Improved Artemia survival rates in tanks receiving ozonated water suggested a positive effect of ozonation on animal health. Although the used ozonation protocol did not hinder biofilm formation, the results suggest ozonation as a promising approach for manipulation of bacterial populations in aquaculture systems, which can prove beneficial for cultured animals.     

Protozoan Predation Is Differentially Affected by Motility of Enteric Pathogens in Water vs. Sediments

Survival of enteric bacteria in aquatic habitats varies depending upon species, strain, and environmental pressures, but the mechanisms governing their fate are poorly understood. Although predation by protozoa is a known, top-down control mechanism on bacterial populations, its influence on the survival of fecal-derived pathogens has not been systematically studied. We hypothesized that motility, a variable trait among pathogens, can influence predation rates and bacterial survival. We compared the survival of two motile pathogens of fecal origin by culturing Escherichia coli O157 and Salmonella enterica Typhimurium. Each species had a motile and non-motile counterpart and was cultured in outdoor microcosms with protozoan predators (Tetrahymena pyriformis) present or absent. Motility had a significant, positive effect on S. enterica levels in water and sediment in the presence or absence of predators. In contrast, motility had a significant negative effect on E. coli O157 levels in sediment, but did not affect water column levels. The presence/absence of protozoa consistently accounted for a greater proportion of the variability in bacterial levels (>95 %) than in bacterial motility (<4 %) in the water column. In sediments, however, motility was more important than predation for both bacteria. Calculations of total CFU/microcosm showed decreasing bacterial concentrations over time under all conditions except for S. enterica in the absence of predation, which increased ～0.5–1.0 log over 5 days. These findings underscore the complexity of predicting the survival of enteric microorganisms in aquatic habitats, which has implications for the accuracy of risk assessment and modeling of water quality.     

Response of performance and bacterial community to oligotrophic stress in biofilm systems for raw water pretreatment

Understanding the dynamics of performance and bacterial community of biofilm under oligotrophic stress is necessary for the process optimization and risk management in biofilm systems for raw water pretreatment. In this study, biofilm obtained from a pilot-scale biofilm reactor was inoculated into a pilot-scale experimental tank for the treatment of oligotrophic raw water. Results showed that the removal of NH₄ ⁺–N was impaired in biofilm systems when influent NH₄ ⁺–N was less than 0.35 mg L^?1 or NH₄ ⁺–N loading rate of less than 7.51 mg L^?1 day^?1. The dominant bacteria detected in biofilm of different carrier were obvious distinct from phylum to genus level under oligotrophic stress. The dominant bacteria in elastic stereo media carrier changed from Proteobacteria (51.1%) to Firmicutes (32.7%), while Proteobacteria was always dominant in suspended ball carrier after long-term operation under oligotrophic conditions. Oligotrophic stress largely decreased the functional bacteria for the removal of nitrogen and organics including many genera in Proteobacteria and Nitrospirae, but increased several genera with spore forming organisms or potential bacterial pathogens in ESM carrier mainly including Bacillus, Mycobacterium, Pseudomonas, etc.     

Kinetics of nitrification in a fixed biofilm reactor using dewatered sludge-fly ash composite ceramic particle as a supporting medium

A mathematical model system was derived to describe the kinetics of ammonium nitrification in a fixed biofilm reactor using dewatered sludge-fly ash composite ceramic particle as a supporting medium. The model incorporates diffusive mass transport and Monod kinetics. The model was solved using a combination of the orthogonal collocation method and Gear’s method. A batch test was conducted to observe the nitrification of ammonium-nitrogen ( \({\text{NH}}_{4}^{ + }\) -N) and the growth of nitrifying biomass. The compositions of nitrifying bacterial community in the batch kinetic test were analyzed using PCR–DGGE method. The experimental results show that the most staining intensity abundance of bands occurred on day 2.75 with the highest biomass concentration of 46.5 mg/L. Chemostat kinetic tests were performed independently to evaluate the biokinetic parameters used in the model prediction. In the column test, the removal efficiency of \({\text{NH}}_{4}^{ + }\) -N was approximately 96 % while the concentration of suspended nitrifying biomass was approximately 16 mg VSS/L and model-predicted biofilm thickness reached up to 0.21 cm in the steady state. The profiles of denaturing gradient gel electrophoresis (DGGE) of different microbial communities demonstrated that indigenous nitrifying bacteria (Nitrospira and Nitrobacter) existed and were the dominant species in the fixed biofilm process.     

A plant growth-promoting rhizobacteria (PGPR) mixture does not display synergistic effects,likely by biofilm but not growth inhibition

Combination of different PGPR strains with complementary characteristics as a mixture to reduce possible instability under fluctuating environment has been considered practical. However, PGPR mixtures do not always play synergistic roles in growth promotion or biological control as reflected in our previous findings and other publications. In this work, we accidentally discovered that a mixture containing two well compatible PGPR strains, Bacillus pumilus WP8 and Erwinia persicinus RA2, did not synergize in growth promotion or biological control of tomato bacterial wilt under field conditions. Considering the importance of PGPR biofilm formation in growth promotion and biocontrol activities, we hypothesized that this phenomenon may be related to inhibition of biofilm formation. In vitro experiments showed that biofilm-formation ability of WP8 was inhibited by both RA2 cells and filtered supernatants collected from RA2 cultures at 12 h (RA2-12) rather than 48 h (RA2-48), even at high-temperatures (within 100°C). An in vivo experiment derived from crystal violet staining yielded similar results. Using liquid chromatography-mass spectrometry (LC-MS), we compared primary and secondary metabolites of RA2 (namely RA2-12 and RA2-48) and found D-glutamine, abundant in RA2-12, as the putative inhibitory factor. Trace amounts of jasmonic acid together with viscous extracellular polysaccharides in RA2-48 likely promoted the rescue of robust biofilm formation. This work suggests that inhibition of biofilm formation should be considered in PGPR mixture development.     

Influence of Lysogeny of Tectiviruses GIL01 and GIL16 on Bacillus thuringiensis Growth,Biofilm Formation,and Swarming Motility

Bacillus thuringiensis is an entomopathogenic bacterium that has been used as an efficient biopesticide worldwide. Despite the fact that this bacterium is usually described as an insect pathogen, its life cycle in the environment is still largely unknown. B. thuringiensis belongs to the Bacillus cereus group of bacteria, which has been associated with many mobile genetic elements, such as species-specific temperate or virulent bacteriophages (phages). Temperate (lysogenic) phages are able to establish a long-term relationship with their host, providing, in some cases, novel ecological traits to the bacterial lysogens. Therefore, this work focuses on evaluating the potential influence of temperate tectiviruses GIL01 and GIL16 on the development of different life traits of B. thuringiensis. For this purpose, a B. thuringiensis serovar israelensis plasmid-cured (nonlysogenic) strain was used to establish bacterial lysogens for phages GIL01 and GIL16, and, subsequently, the following life traits were compared among the strains: kinetics of growth, metabolic profiles, antibiotics susceptibility, biofilm formation, swarming motility, and sporulation. The results revealed that GIL01 and GIL16 lysogeny has a significant influence on the bacterial growth, sporulation rate, biofilm formation, and swarming motility of B. thuringiensis. No changes in metabolic profiles or antibiotic susceptibilities were detected. These findings provide evidence that tectiviruses have a putative role in the B. thuringiensis life cycle as adapters of life traits with ecological advantages.     

Antimicrobial and Antibiofilm Activity of Mauriporin,a Multifunctional Scorpion Venom Peptide

Many pathogenic free living and biofilm forming bacterial organisms can cause serious infections to humans that could consequently have devastating effects on human health. A significant number of these microbial organisms are resistant to almost all known conventional antibiotics and the ability of some these strains to form sessile communities of biofilms increases the resistance ability of bacteria to antibiotic treatment. Global research is currently focused on finding novel therapies to counteract the threat of bacterial and biofilm infections rather than using conventional antibiotics. Mauriporin, a novel cationic α-helical peptide identified from the venom derived cDNA library of the scorpion Androctonus mauritanicus was reported to display selective cytotoxic and anti-proliferative activity against prostate cancer cell lines. In the present study, we investigated the antimicrobial and antibiofilm activities of Mauriporin. Our results show that Mauriporin displays potent antimicrobial activities against a range of Gram-positive and Gram-negative planktonic bacteria with MIC values in the range 5 µM to 10 µM. Mauriporin was also able to prevent Pseudomonas aeruginosa biofilm formation while showing weak hemolytic activity towards human erythrocytes. Studies on the mechanism of action of Mauriporin revealed that the peptide is probably inducing bacterial cell death through membrane permeabilization determined by the release of β-galactosidase enzyme from peptide treated Escherichia coli cells. Moreover, DNA binding studies found that Mauriporin can cause potent binding to intracellular DNA. All these results indicate that Mauriporin has a considerable potential for therapeutic application as a novel drug candidate for eradicating bacterial infections.     

Phage-host associations in a full-scale activated sludge plant during sludge bulking

Sludge bulking, a notorious microbial issue in activated sludge plants, is always accompanied by dramatic changes in the bacterial community. Despite large numbers of phages in sludge systems, their responses to sludge bulking and phage-host associations during bulking are unknown. In this study, high-throughput sequencing of viral metagenomes and bacterial 16S rRNA genes were employed to characterize viral and bacterial communities in a sludge plant under different sludge conditions (sludge volume index (SVI) of 180, 132, and 73 ml/g). Bulking sludges (SVI > 125 ml/g) taken about 10 months apart exhibited similar bacterial and viral composition. This reflects ecological resilience of the sludge microbial community and indicates that changes in viral and bacterial populations correlate closely with each other. Overgrowth of “Candidatus Microthrix parvicella” led to filamentous bulking, but few corresponding viral genotypes were identified. In contrast, sludge viromes were characterized by numerous contigs associated with “Candidatus Accumulibacter phosphatis,” suggesting an abundance of corresponding phages in the sludge viral community. Notably, while nitrifiers (mainly Nitrosomonadaceae and Nitrospiraceae) declined significantly along with sludge bulking, their corresponding viral contigs were identified more frequently and with greater abundance in the bulking viromes, implying that phage-mediated lysis might contribute to the loss of autotrophic nitrifiers under bulking conditions.     

Identification and Characterization of a Phosphodiesterase That Inversely Regulates Motility and Biofilm Formation in Vibrio cholerae

Vibrio cholerae switches between free-living motile and surface-attached sessile lifestyles. Cyclic diguanylate (c-di-GMP) is a signaling molecule controlling such lifestyle changes. C-di-GMP is synthesized by diguanylate cyclases (DGCs) that contain a GGDEF domain and is degraded by phosphodiesterases (PDEs) that contain an EAL or HD-GYP domain. We constructed in-frame deletions of all V. cholerae genes encoding proteins with GGDEF and/or EAL domains and screened mutants for altered motility phenotypes. Of 52 mutants tested, four mutants exhibited an increase in motility, while three mutants exhibited a decrease in motility. We further characterized one mutant lacking VC0137 (cdgJ), which encodes an EAL domain protein. Cellular c-di-GMP quantifications and in vitro enzymatic activity assays revealed that CdgJ functions as a PDE. The cdgJ mutant had reduced motility and exhibited a small decrease in flaA expression; however, it was able to produce a flagellum. This mutant had enhanced biofilm formation and vps gene expression compared to that of the wild type, indicating that CdgJ inversely regulates motility and biofilm formation. Genetic interaction analysis revealed that at least four DGCs, together with CdgJ, control motility in V. cholerae.Cyclic diguanylate (c-di-GMP) is a ubiquitous second messenger in bacteria. It is synthesized by diguanylate cyclases (DGCs) that contain a GGDEF domain and is degraded by phosphodiesterases (PDEs) that contain an EAL or HD-GYP domain (46, 48, 50). The receptors of c-di-GMP, which can be proteins or RNAs (riboswitches), bind to c-di-GMP and subsequently transmit the signal to downstream targets (22). C-di-GMP signaling is predicted to occur via a common or localized c-di-GMP pool(s) through so-called c-di-GMP signaling modules harboring DGCs and PDEs, receptors, and targets that affect cellular function (22).C-di-GMP controls various cellular functions, including the transition between a planktonic lifestyle and biofilm lifestyle. In general, high concentrations of c-di-GMP promote the expression of adhesive matrix components and result in biofilm formation, while low concentrations of c-di-GMP result in altered motility upon changes in flagellar or pili function and/or production (reviewed in reference 25). C-di-GMP inversely regulates motility and biofilm formation by implementing control at different levels through gene expression or through posttranslational mechanisms (reviewed in reference 25).Vibrio cholerae, the causative agent of the disease cholera, uses c-di-GMP signaling to undergo a motile-to-sessile lifestyle switch that is important for both environmental and in vivo stages of the V. cholerae life cycle. The survival of the pathogen in both natural aquatic environments and during infection depends on the appropriate regulation of motility, surface attachment, and colonization factors (26). The V. cholerae genome encodes a total of 62 putative c-di-GMP metabolic enzymes: 31 with a GGDEF domain, 12 with an EAL domain, 10 with both GGDEF and EAL domains, and 9 with an HD-GYP domain (21). V. cholerae contains a few known or predicted c-di-GMP receptors: two riboswitches (53), five PilZ domain proteins (43), VpsT (31), and CdgG (6). C-di-GMP regulates virulence, motility, biofilm formation, and the smooth-to-rugose phase variation in V. cholerae (6, 8, 9, 12, 30, 33, 43, 45, 54, 56, 57). However, particular sets of proteins have not been matched to discrete cellular processes.Some of the DGCs and PDEs involved in regulating motility in V. cholerae have been identified: rocS and cdgG mutants exhibit a decrease in motility (45), while cdgD and cdgH mutants exhibit an increase in motility (6). In addition, VieA (PDE) positively regulates motility in the V. cholerae classical biotype but not in the El Tor biotype (7). AcgA (PDE) positively regulates motility at low concentrations of inorganic phosphate (42). In this study, we investigated the role of each putative gene encoding DGCs and PDEs in controlling cell motility. In addition to the already-characterized proteins CdgD, CdgH, and RocS, we identified two putative DGCs (CdgK and CdgL) that negatively control motility and a putative PDE (CdgJ) that positively controls motility. We further characterized CdgJ and showed that it functions as a PDE and inversely regulates motility and biofilm formation. Genetic interaction studies revealed that DGCs CdgD, CdgH, CdgL, and CdgK and PDE CdgJ form a c-di-GMP signaling network to control motility in V. cholerae.     

Increase in Rhamnolipid Synthesis under Iron-Limiting Conditions Influences Surface Motility and Biofilm Formation in Pseudomonas aeruginosa

Iron is an essential element for life but also serves as an environmental signal for biofilm development in the opportunistic human pathogen Pseudomonas aeruginosa. Under iron-limiting conditions, P. aeruginosa displays enhanced twitching motility and forms flat unstructured biofilms. In this study, we present evidence suggesting that iron-regulated production of the biosurfactant rhamnolipid is important to facilitate the formation of flat unstructured biofilms. We show that under iron limitation the timing of rhamnolipid expression is shifted to the initial stages of biofilm formation (versus later in biofilm development under iron-replete conditions) and results in increased bacterial surface motility. In support of this observation, an rhlAB mutant defective in biosurfactant production showed less surface motility under iron-restricted conditions and developed structured biofilms similar to those developed by the wild type under iron-replete conditions. These results highlight the importance of biosurfactant production in determining the mature structure of P. aeruginosa biofilms under iron-limiting conditions.The biofilm mode of bacterial growth is a surface-attached state in which cells are closely packed and encased in an extracellular polymeric matrix (10, 27). Biofilms are abundant in nature and are of clinical, environmental, and industrial importance. Biofilm development is known to follow a series of complex but discrete and tightly regulated steps (18, 27), including (i) microbial attachment to the surface, (ii) growth and aggregation of cells into microcolonies, (iii) maturation, and (iv) dissemination of progeny cells that can colonize new niches. Over the last decade, several key processes important for biofilm formation have been identified, including quorum sensing (12) and surface motility (28).One of the best-studied model organisms for biofilm development is the bacterium Pseudomonas aeruginosa (10), a notorious opportunistic pathogen which causes many types of infections, including biofilm-associated chronic lung infections in individuals with cystic fibrosis (10, 24, 41). Like most organisms, P. aeruginosa requires iron for growth, as iron serves as a cofactor for enzymes that are involved in many basic cellular functions and metabolic pathways. Recent work has shown that at iron concentrations that are not limiting for growth, this metal serves as a signal for biofilm development (40). Iron limitation imposed, for example, by the mammalian iron chelator lactoferrin blocks the ability of P. aeruginosa biofilms to mature from thin layers of cells attached to a surface into large multicellular mushroom-like biofilm structures (40). By chelating iron, lactoferrin induces twitching motility (a specialized form of surface motility), which causes the cells to move across the surface instead of settling down to form structured communities (39, 40). In a recent paper, Berlutti et al. (5) provided further support for the role of iron in cell aggregation and biofilm formation. They reported that in the liquid phase, iron limitation induced motility and transition to the free-living (i.e., planktonic) mode of growth, while increased iron concentrations facilitated cell aggregation and biofilm formation. We recently demonstrated that iron limitation-induced twitching motility is regulated by quorum sensing (31). Quorum sensing allows bacteria to sense and respond to their population density via the production of small diffusible signal molecules. In P. aeruginosa and many other Gram-negative bacteria, these signal molecules are N-acyl homoserine lactones (acyl-HSLs), which have specific receptors (R proteins) (16, 30). P. aeruginosa possesses two acyl-HSL quorum-sensing systems, one for production of and response to N-3-oxo-dodecanoyl homoserine lactone (3OC₁₂-HSL) (LasR-LasI) and the other for production of and response to N-butanoyl homoserine lactone (C₄-HSL) (RhlR-RhlI) (35, 37). We have reported that an rhlI mutant unable to synthesize the C₄-HSL signal was impaired in iron limitation-induced twitching motility and formed structured biofilms under iron-limiting conditions (31).The correlation between twitching motility, the RhlR-RhlI quorum-sensing system, and iron-regulated biofilm formation led us to hypothesize that rhamnolipids are involved in mediating this process. Rhamnolipids are surface-active amphipathic molecules composed of a hydrophobic lipid and a hydrophilic sugar moiety and compose the main constituents of the biosurfactant produced by P. aeruginosa (reviewed in reference 42). The biosurfactant is required for a form of surface motility called swarming, where it functions as a wetting agent and reduces surface tension (8, 14). Furthermore, elements constituting the biosurfactant were recently shown to modulate the swarming behavior by acting as chemotactic-like stimuli (43). Rhamnolipids are also important in maintaining biofilm structure and inducing biofilm dispersion (6, 11, 29). Their synthesis requires the expression of the rhlAB operon, which is regulated by the RhlR-RhlI quorum-sensing system (14, 25, 32) and is also induced under iron-limiting conditions (14).In this study, we test this hypothesis and demonstrate that rhamnolipid production is induced under iron-limiting conditions and that this promotes twitching motility. We found that increased expression of rhamnolipid synthesis genes during early biofilm development under iron-limiting conditions induces surface motility and results in formation of a thin flat biofilm. Furthermore, a mutant that is incapable of synthesizing rhamnolipids does not display twitching motility under iron-limiting conditions and thus forms structured biofilms under these conditions. These results highlight the importance of biosurfactant production in determining the architecture of mature P. aeruginosa biofilms under iron-limiting conditions.     

In Vitro Antibacterial Activity of a Novel Resin-Based Pulp Capping Material Containing the Quaternary Ammonium Salt MAE-DB and Portland Cement

Background

Vital pulp preservation in the treatment of deep caries is challenging due to bacterial infection. The objectives of this study were to synthesize a novel, light-cured composite material containing bioactive calcium-silicate (Portland cement, PC) and the antimicrobial quaternary ammonium salt monomer 2-methacryloxylethyl dodecyl methyl ammonium bromide (MAE-DB) and to evaluate its effects on Streptococcus mutans growth in vitro.

Methods

The experimental material was prepared from a 2∶1 ratio of PC mixed with a resin of 2-hydroxyethylmethacrylate, bisphenol glycerolate dimethacrylate, and triethylene glycol dimethacrylate (4∶3∶1) containing 5 wt% MAE-DB. Cured resin containing 5% MAE-DB without PC served as the positive control material, and resin without MAE-DB or PC served as the negative control material. Mineral trioxide aggregate (MTA) and calcium hydroxide (Dycal) served as commercial controls. S. mutans biofilm formation on material surfaces and growth in the culture medium were tested according to colony-forming units (CFUs) and metabolic activity after 24 h incubation over freshly prepared samples or samples aged in water for 6 months. Biofilm formation was also assessed by Live/Dead staining and scanning electron microscopy.

Results

S. mutans biofilm formation on the experimental material was significantly inhibited, with CFU counts, metabolic activity, viability staining, and morphology similar to those of biofilms on the positive control material. None of the materials affected bacterial growth in solution. Contact-inhibition of biofilm formation was retained by the aged experimental material. Significant biofilm formation was observed on MTA and Dycal.

Conclusion

The synthesized material containing HEMA-BisGMA-TEGDMA resin with MAE-DB as the antimicrobial agent and PC to support mineralized tissue formation inhibited S. mutans biofilm formation even after aging in water for 6 months, but had no inhibitory effect on bacteria in solution. Therefore, this material shows promise as a pulp capping material for vital pulp preservation in the treatment of deep caries.     

Cyclic-di-GMP-Mediated Repression of Swarming Motility by Pseudomonas aeruginosa: the pilY1 Gene and Its Impact on Surface-Associated Behaviors

The intracellular signaling molecule cyclic-di-GMP (c-di-GMP) has been shown to influence surface-associated behaviors of Pseudomonas aeruginosa, including biofilm formation and swarming motility. Previously, we reported a role for the bifA gene in the inverse regulation of biofilm formation and swarming motility. The bifA gene encodes a c-di-GMP-degrading phosphodiesterase (PDE), and the ΔbifA mutant exhibits increased cellular pools of c-di-GMP, forms hyperbiofilms, and is unable to swarm. In this study, we isolated suppressors of the ΔbifA swarming defect. Strains with mutations in the pilY1 gene, but not in the pilin subunit pilA gene, show robust suppression of the swarming defect of the ΔbifA mutant, as well as its hyperbiofilm phenotype. Despite the ability of the pilY1 mutation to suppress all the c-di-GMP-related phenotypes, the global pools of c-di-GMP are not detectably altered in the ΔbifA ΔpilY1 mutant relative to the ΔbifA single mutant. We also show that enhanced expression of the pilY1 gene inhibits swarming motility, and we identify residues in the putative VWA domain of PilY1 that are important for this phenotype. Furthermore, swarming repression by PilY1 specifically requires the diguanylate cyclase (DGC) SadC, and epistasis analysis indicates that PilY1 functions upstream of SadC. Our data indicate that PilY1 participates in multiple surface behaviors of P. aeruginosa, and we propose that PilY1 may act via regulation of SadC DGC activity but independently of altering global c-di-GMP levels.Pseudomonas aeruginosa forms surface-attached communities known as biofilms, and this microbe is also capable of surface-associated motility, including twitching and swarming. The mechanism by which cells regulate and coordinate these various surface-associated behaviors, or how these microbes transition from one surface behavior to another, has yet to be elucidated. Given that P. aeruginosa is capable of such diverse surface-associated lifestyles, this Gram-negative organism serves as a useful model to address questions regarding the regulation of surface-associated behaviors.Recent studies indicate that biofilm formation and swarming motility by P. aeruginosa are inversely regulated via a common pathway (12, 27, 37). Important factors that influence early biofilm formation by P. aeruginosa strain PA14 include control of flagellar motility and the robust production of the Pel exopolysaccharide (EPS). Swarming occurs when cells move across a hydrated, viscous semisolid surface, and like biofilm formation, flagellar function is important for this surface-associated motility. Additionally, swarming requires production of rhamnolipid surfactant acting as a surface-wetting agent (25, 58). In contrast to biofilm formation, swarming motility is enhanced in strains which are defective for the production of Pel EPS (12).The inverse regulation of biofilm formation and swarming motility is reminiscent of the regulation of sessile and motile behaviors that occurs in a wide range of bacterial species via the intracellular signaling molecule cyclic-di-GMP (c-di-GMP) (17, 24, 50, 51, 56). High levels of this signaling molecule promote sessile behaviors and inhibit motility, whereas low levels of c-di-GMP favor motile behaviors (8, 9, 22, 56). Recently, we reported that the BifA phosphodiesterase, which catalyzes the breakdown of c-di-GMP, inversely regulates biofilm formation and swarming motility (27). In addition, Merritt et al. reported that SadC, a diguanylate cyclase (DGC) which synthesizes c-di-GMP, participates with BifA to modulate cellular c-di-GMP levels and thus regulate biofilm formation and swarming motility (37).Consistent with a role for BifA as a c-di-GMP phosphodiesterase, ΔbifA mutants exhibit increased cellular pools of c-di-GMP relative to the wild type (WT) (27). Phenotypically, ΔbifA mutants form hyperbiofilms and are unable to swarm. The hyperbiofilm phenotype of the ΔbifA mutant results largely from increased synthesis of the pel-derived polysaccharide; that is, the ΔbifAΔpel double mutant shows a marked decrease in biofilm formation compared to the ΔbifA mutant (27). Interestingly, elevated Pel polysaccharide production alone is not sufficient to explain the swarming defect of the ΔbifA mutant, as the ΔbifAΔpel double mutant recovers only minimal swarming ability (27). These data indicate that high levels of c-di-GMP inhibit swarming motility in a largely Pel-independent manner.To better understand how elevated c-di-GMP levels in the cell inhibit swarming motility, we exploited the swarming defect of the ΔbifA mutant, and using a genetic screen, we identified suppressors in the ΔbifA background that restored the ability to swarm. Here we report a role for the PilY1 protein in repression of swarming motility in the ΔbifA mutant background. Our data are consistent with a model in which PilY1 functions upstream of the c-di-GMP diguanylate cyclase SadC to regulate swarming motility by P. aeruginosa.     

Bacteriophage Diversity in the North Sea

In recent years interest in bacteriophages in aquatic environments has increased. Electron microscopy studies have revealed high numbers of phage particles (10⁴ to 10⁷ particles per ml) in the marine environment. However, the ecological role of these bacteriophages is still unknown, and the role of the phages in the control of bacterioplankton by lysis and the potential for gene transfer are disputed. Even the basic questions of the genetic relationships of the phages and the diversity of phage-host systems in aquatic environments have not been answered. We investigated the diversity of 22 phage-host systems after 85 phages were collected at one station near a German island, Helgoland, located in the North Sea. The relationships among the phages were determined by electron microscopy, DNA-DNA hybridization, and host range studies. On the basis of morphology, 11 phages were assigned to the virus family Myoviridae, 7 phages were assigned to the family Siphoviridae, and 4 phages were assigned to the family Podoviridae. DNA-DNA hybridization confirmed that there was no DNA homology between phages belonging to different families. We found that the 22 marine bacteriophages belonged to 13 different species. The host bacteria were differentiated by morphological and physiological tests and by 16S ribosomal DNA sequencing. All of the bacteria were gram negative, facultatively anaerobic, motile, and coccoid. The 16S rRNA sequences of the bacteria exhibited high levels of similarity (98 to 99%) with the sequences of organisms belonging to the genus Pseudoalteromonas, which belongs to the γ subdivision of the class Proteobacteria.The marine bacterial community is responsible for a considerable portion of primary production and regeneration of nutrients in the microbial loop and is associated with a great variety of marine bacteriophages (5, 12). These phages are capable of infecting a large portion of the bacterioplankton (32, 34). It is assumed that as part of the marine food web, bacteriophages play important quantitative and qualitative roles in controlling marine bacterial populations (8, 24, 34, 39, 45). The phenotypic diversity and genotypic diversity of the phage populations are related to the interaction between phages and their host organisms, which provides a tool for understanding the interaction itself (13). To estimate the influence of marine bacteriophages on the diversity of bacterioplankton, we investigated phage diversity. The virus species concept proposed by Murphy et al. (37) delineates seven different families of bacteriophages based on morphological criteria and provides criteria for new phage species based on several traits, such as DNA homologies, serological data, protein profiles, and host ranges.In this paper, we describe the diversity and genetic relationships of marine phages based on investigations of 22 representatives from 85 phage-host systems (35, 36) collected between 1988 and 1992 from waters around an island, Helgoland, located in the North Sea. All of the phages were virulent and formed plaques on their host bacteria. We assigned the phages to different virus families, species, and strains based on morphology, DNA homology, and host range. Furthermore, we characterized the phenotypic and genotypic features of the host bacteria.     

Effects of antimicrobial peptide L-K6, a temporin-1CEb analog on oral pathogen growth,Streptococcus mutans biofilm formation,and anti-inflammatory activity

Dental caries and periodontitis are common bacterial mouth infections. As a potentially attractive substitute for conventional antibiotics, antimicrobial peptides have been widely tested and used for controlling bacterial infections. In this study, we tested the efficacy of the peptides from the skin secretions of Rana chensinensis for killing several major cariogenic and periodontic pathogens as well as Candida albicans. L-K6, a temporin-1CEb analog, exhibited high antimicrobial activity against the tested oral pathogens and was able to inhibit Streptococcus mutans biofilm formation and reduce 1-day-old S. mutans biofilms with a minimum biofilm inhibitory concentration and reducing concentration of 3.13 and 6.25 μM, respectively. The results of confocal laser scanning microscopy demonstrated that the peptide significantly reduced cell viability within oral biofilms. Furthermore, as little as 5 μM L-K6 significantly inhibited lipopolysaccharide (LPS)- and interleukin-1β-induced productions of interleukin-8 and tumor necrosis factor-α from THP-1 monocytic cells. This anti-inflammatory activity is associated with the binding of L-K6 to LPS and neutralizing LPS-induced proinflammatory responses in THP-1 cells, as well as dissociating LPS aggregates. Our results suggest that L-K6 may have potential clinical applications in treating dental caries by killing S. mutans within dental plaque and acting as anti-inflammatory agents in infected tissues.     

Biofilms

The ability to form biofilms is a universal attribute of bacteria. Biofilms are multicellular communities held together by a self-produced extracellular matrix. The mechanisms that different bacteria employ to form biofilms vary, frequently depending on environmental conditions and specific strain attributes. In this review, we emphasize four well-studied model systems to give an overview of how several organisms form biofilms: Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus. Using these bacteria as examples, we discuss the key features of biofilms as well as mechanisms by which extracellular signals trigger biofilm formation.Bacteria are able to grow adhered to almost every surface, forming architecturally complex communities termed biofilms. In biofilms, cells grow in multicellular aggregates that are encased in an extracellular matrix produced by the bacteria themselves (Branda et al. 2005; Hall-Stoodley and Stoodley 2009). Biofilms impact humans in many ways as they can form in natural, medical, and industrial settings. For instance, formation of biofilms on medical devices, such as catheters or implants often results in difficult-to-treat chronic infections (Hall-Stoodley et al. 2004; Donlan 2008; Hatt and Rather 2008). Moreover, infections have been associated with biofilm formation on human surfaces such as teeth, skin, and the urinary tract (Hatt and Rather 2008). However, biofilms on human surfaces are not always detrimental. For example, dental plaque biofilms comprise dozens of species and the community composition frequently determines the presence or absence of disease. In dental plaque, there is a progression of colonization and the presence of beneficial species antagonizes colonization by detrimental organisms (Kreth et al. 2008). But biofilms form everywhere. For example, biofilms form on the hulls of ships and inside pipes where they cause severe problems (de Carvalho 2007). On the other hand, in many natural settings, biofilm formation often allows mutualistic symbioses. For instance, Actinobacteria often grow on ants, allowing the ants to maintain pathogen-free fungal gardens (Currie 2001; Danhorn and Fuqua 2007). Given the vast potential benefits and detriments that biofilms can confer, it is essential that we understand how bacteria thrive in these communities.There are numerous benefits that a bacterial community might obtain from the formation of biofilms. Biofilms confer resistance to many antimicrobials, protection from protozoan grazing, and protection against host defenses (Mah and O’Toole 2001; Matz and Kjelleberg 2005; Anderson and O’Toole 2008). One possible reason for the increased resistance to environmental stresses observed in biofilm cells appears to be the increase in the portion of persister cells within the biofilm (Lewis 2005). Despite being genetically identical to the rest of the population, persister cells are resistant to many antibiotics and are nondividing. Persister cells have been proposed to be protected from the action of antibiotics because they express toxin–antitoxin systems where the target of the antibiotics is blocked by the toxin modules (Lewis 2005). In addition to an increase in persisters, the presence of an extracellular matrix protects constituent cells from external aggressions. Extracellular matrices also act as a diffusion barrier to small molecules (Anderson and O’Toole 2008; Hall-Stoodley and Stoodley 2009). Related to this, in biofilms the diffusion of nutrients, vitamins, or cofactors is slower resulting in a bacterial community in which some of cells are metabolically inactive. Furthermore, the rate of bacterial growth is influenced by the fact that cells within a biofilm are confined to a limited space (Stewart and Franklin 2008). This condition is similar to the stationary phase created in laboratory conditions. Hence, biofilm formation in a way represents the natural stationary phase of bacterial growth. During stationary phase, bacteria profoundly change their physiology by increasing production of secondary metabolites such as antibiotics, pigments, and other small-molecules (Martin and Liras 1989). These secondary metabolites also function as signaling molecules to initiate the process of biofilm formation or to inhibit biofilm formation by other organisms that inhabit the same habitat (Lopez and Kolter 2009). In this article, we review the metabolic processes that characterize biofilm formation for a handful of well-studied bacterial organisms: Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and Bacillus subtilis. In addition, we address the function of secondary metabolites and their role as signaling molecules during biofilm formation.