Intracellular offspring released from SFB filaments are flagellated.

The gut commensal segmented filamentous bacterium (SFB) attaches to the ileal epithelium and potently stimulates the host immune system. Using transmission electron microscopy (TEM), we show that mouse and rat SFB are flagellated above the concave tip at the unicellular intracellular offspring (IO) stage and that flagellation occurs prior to full IO differentiation and release of IOs from SFB filaments. This finding adds a missing link to the SFB life cycle.

Effects of sub-minimal inhibitory concentrations of antibiotics on the morphology and surface hydrophobicity of periodontopathic anaerobes.

It has been reported that sub-minimal inhibitory concentrations (sub-MICs) of antibiotics are capable of altering bacterial surface properties and phenotype. In this study, the effects of sub-MICs of certain antibiotics on surface hydrophobicity, cell morphology, and protein profile were ascertained using Fusobacterium nucleatum, Porphyromonas gingivalis and Treponema denticola strains, which are pathogenic bacterial species in periodontal diseases. The MICs of antibiotics were determined by culturing bacteria in media supplemented with serially diluted antibiotic solutions, and sub-MIC of antibiotics was used. The effect of sub-MIC of antibiotics on cell morphology was determined by scanning electron microscopy. Microscopic observation of F. nucleatum and P. gingivalis grown at a sub-MIC of amoxicillin revealed cell enlargement. T. denticola grown at a sub-MIC of doxycycline also showed cell elongation. The relative surface hydrophobicity determined by measuring the ability of the bacteria to absorb n-hexadecane revealed an increase in surface hydrophobicity of F. nucleatum grown at sub-MIC of penicillin and amoxicillin, but a decrease with metronidazole; whereas increased hydrophobicity was observed in T. denticola grown at sub-MIC of doxycycline, metronidazole and tetracycline. The surface hydrophobicity of P. gingivalis increased only when grown in sub-MIC of metronidazole. The protein expression profile of the treated bacteria differed from their respective controls. These results confirmed that sub-MIC concentrations of antibiotics can affect the phenotype, surface properties and morphology of periodontal pathogenic anaerobic bacteria.

Anaerobic consortia of fungi and sulfate reducing bacteria in deep granite fractures.

The deep biosphere is one of the least understood ecosystems on Earth. Although most microbiological studies in this system have focused on prokaryotes and neglected microeukaryotes, recent discoveries have revealed existence of fossil and active fungi in marine sediments and sub-seafloor basalts, with proposed importance for the subsurface energy cycle. However, studies of fungi in deep continental crystalline rocks are surprisingly few. Consequently, the characteristics and processes of fungi and fungus-prokaryote interactions in this vast environment remain enigmatic. Here we report the first findings of partly organically preserved and partly mineralized fungi at great depth in fractured crystalline rock (-740 m). Based on environmental parameters and mineralogy the fungi are interpreted as anaerobic. Synchrotron-based techniques and stable isotope microanalysis confirm a coupling between the fungi and sulfate reducing bacteria. The cryptoendolithic fungi have significantly weathered neighboring zeolite crystals and thus have implications for storage of toxic wastes using zeolite barriers.Deep subsurface microorganisms play an important role in nutrient cycling, yet little is known about deep continental fungal communities. Here, the authors show organically preserved and partly mineralized fungi at 740 m depth, and find evidence of an anaerobic fungi and sulfate reducing bacteria consortium.

Support material dictates the attached biomass characteristics during the immobilization process in anaerobic continuous-flow packed-bed bioreactor.

Hydrogen is considered to be an ideal energy alternative to replace environmentally burdensome fossil fuels. For its long-term production the immobilized biofilm system is the most promising and to choose the right support material the most challenging. In this respect, the anaerobic up-flow bioreactors packed with four most used support materials (polyethylene, polyurethane, activated carbon and expanded clay) were tested to investigate the crucial bacteria sensitive period-the immobilization process. Seven-day-operation was necessary and sufficient to reach metabolic and microbial stability regardless of support material used. The support material had an influence on the microbial metabolic activity as well as on quantity and quality characteristics of the immobilized microbial community, being polyethylene and expanded clay more appropriate as supports among the materials evaluated; this could be attributed to pH alteration. The obtained results suggest that the support material dictates the outcome of the immobilization process in the anaerobic continuous-flow bioreactor.

Illuminating vital surface molecules of symbionts in health and disease.

The immunomodulatory surface molecules of commensal and pathogenic bacteria are critical to microorganisms' survival and the host's response1,2. Recent studies have highlighted the unique and important responses elicited by commensal-derived surface macromolecules3-5. However, the technology available to track these molecules in host cells and tissues remains primitive. We report, here, an interdisciplinary approach that uses metabolic labelling combined with bioorthogonal click chemistry (that is, reactions performed in living organisms)6 to specifically tag up to three prominent surface immunomodulatory macromolecules-peptidoglycan, lipopolysaccharide and capsular polysaccharide-either simultaneously or individually in live anaerobic commensal bacteria. Importantly, the peptidoglycan labelling enables, for the first time, the specific labelling of live endogenous, anaerobic bacteria within the mammalian host. This approach has allowed us to image and track the path of labelled surface molecules from live, luminal bacteria into specific intestinal immune cells in the living murine host during health and disease. The chemical labelling of three specific macromolecules within a live organism offers the potential for in-depth visualization of host-pathogen interactions.

Anaerobes in biofilm-based healthcare-associated infections.

Anaerobic bacteria can cause an infection when they encounter a permissive environment within the host. These opportunistic pathogens are seldom recovered as single isolates but more frequently are involved in polymicrobial infections, together with other anaerobes or aerobes. Nowadays it's known that some anaerobic bacteria are also able to grow as biofilm even if this feature and its role in the healthcare-associated infections (HAIs) are still poorly characterized. As consequence, the involvement of biofilm-forming anaerobic bacteria in infections related to healthcare procedures, including surgery and medical devices implantation, is underestimated.The current knowledge on the role of biofilm-growing anaerobes in HAIs has been here reviewed, with particular reference to respiratory, intestinal, intra-abdominal, wound, and urogenital tract infections. Even if the data are still scarce, the ability to form biofilm of opportunistic anaerobic species and their possible role as causative agents of HAIs should alert even more clinicians and microbiologists on the need to search for anaerobes in clinical samples when their presence can be reasonably assumed.

Microbial community composition and ultrastructure of granules from a full-scale anammox reactor.

Granules in anammox reactors contain besides anammox bacteria other microbial communities whose identity and relationship with the anammox bacteria are not well understood. High calcium concentrations are often supplied to anammox reactors to obtain sufficient bacterial aggregation and biomass retention. The aim of this study was to provide the first characterization of bacterial and archaeal communities in anammox granules from a full-scale anammox reactor and to explore on the possible role of calcium in such aggregates. High magnification imaging using backscattered electrons revealed that anammox bacteria may be embedded in calcium phosphate precipitates. Pyrosequencing of 16S rRNA gene fragments showed, besides anammox bacteria (Brocadiacea, 32%), substantial numbers of heterotrophic bacteria Ignavibacteriacea (18%) and Anaerolinea (7%) along with heterotrophic denitrifiers Rhodocyclacea (9%), Comamonadacea (3%), and Shewanellacea (3%) in the granules. It is hypothesized that these bacteria may form a network in which heterotrophic denitrifiers cooperate to achieve a well-functioning denitrification system as they can utilize the nitrate intrinsically produced by the anammox reaction. This network may provide a niche for the proliferation of archaea. Hydrogenotrophic methananogens, which scavenge the key fermentation product H2, were the most abundant archaea detected. Cells resembling the polygon-shaped denitrifying methanotroph Candidatus Methylomirabilis oxyfera were observed by electron microscopy. It is hypothesized that the anammox process in a full-scale reactor triggers various reactions overall leading to efficient denitrification and a sink of carbon as biomass in anammox granules.

Microbiology of explanted suture segments from infected and noninfected surgical patients.

Sutures under selective host/environmental factors can potentiate postoperative surgical site infection (SSI). The present investigation characterized microbial recovery and biofilm formation from explanted absorbable (AB) and nonabsorbable (NAB) sutures from infected and noninfected sites. AB and NAB sutures were harvested from noninfected (70.9%) and infected (29.1%) sites in 158 patients. At explantation, devices were sonicated and processed for qualitative/quantitative bacteriology; selective sutures were processed for scanning electron microscopy (SEM). Bacteria were recovered from 85 (53.8%) explanted sites; 39 sites were noninfected, and 46 were infected. Suture recovery ranged from 11.1 to 574.6 days postinsertion. A significant difference in mean microbial recovery between noninfected (1.2 isolates) and infected (2.7 isolates) devices (P < 0.05) was noted. Staphylococcus epidermidis, Staphylococcus aureus, coagulase-negative staphylococci (CNS), Peptostreptococcus spp., Bacteroides fragilis, Escherichia coli, Enterococcus spp., Pseudomonas aeruginosa, and Serratia spp. were recovered from infected devices, while commensal skin flora was recovered from noninfected devices. No significant difference in quantitative microbial recovery between infected monofilament and multifilament sutures was noted. Biofilm was present in 100% and 66.6% of infected and noninfected devices, respectively (P < 0.042). We conclude that both monofilament and braided sutures provide a hospitable surface for microbial adherence: (i) a significant difference in microbial recovery from infected and noninfected sutures was noted, (ii) infected sutures harbored a mixed flora, including multidrug-resistant health care-associated pathogens, and (iii) a significant difference in the presence or absence of a biofilm in infected versus noninfected explanted devices was noted. Further studies to document the benefit of focused risk reduction strategies to minimize suture contamination and biofilm formation postimplantation are warranted.

Mimicking the oxygen minimum zones: stimulating interaction of aerobic archaeal and anaerobic bacterial ammonia oxidizers in a laboratory-scale model system.

In marine oxygen minimum zones (OMZs), ammonia-oxidizing archaea (AOA) rather than marine ammonia-oxidizing bacteria (AOB) may provide nitrite to anaerobic ammonium-oxidizing (anammox) bacteria. Here we demonstrate the cooperation between marine anammox bacteria and nitrifiers in a laboratory-scale model system under oxygen limitation. A bioreactor containing 'Candidatus Scalindua profunda' marine anammox bacteria was supplemented with AOA (Nitrosopumilus maritimus strain SCM1) cells and limited amounts of oxygen. In this way a stable mixed culture of AOA, and anammox bacteria was established within 200 days while also a substantial amount of endogenous AOB were enriched. 'Ca. Scalindua profunda' and putative AOB and AOA morphologies were visualized by transmission electron microscopy and a C18 anammox [3]-ladderane fatty acid was highly abundant in the oxygen-limited culture. The rapid oxygen consumption by AOA and AOB ensured that anammox activity was not affected. High expression of AOA, AOB and anammox genes encoding for ammonium transport proteins was observed, likely caused by the increased competition for ammonium. The competition between AOA and AOB was found to be strongly related to the residual ammonium concentration based on amoA gene copy numbers. The abundance of archaeal amoA copy numbers increased markedly when the ammonium concentration was below 30 µM finally resulting in almost equal abundance of AOA and AOB amoA copy numbers. Massive parallel sequencing of mRNA and activity analyses further corroborated equal abundance of AOA and AOB. PTIO addition, inhibiting AOA activity, was employed to determine the relative contribution of AOB versus AOA to ammonium oxidation. The present study provides the first direct evidence for cooperation of archaeal ammonia oxidation with anammox bacteria by provision of nitrite and consumption of oxygen.

Anaerobic ammonium-oxidizing bacteria: unique microorganisms with exceptional properties.

Anaerobic ammonium-oxidizing (anammox) bacteria defy many microbiological concepts and share numerous properties with both eukaryotes and archaea. Among their most intriguing characteristics are their compartmentalized cell plan and archaeon-like cell wall. Here we review our current knowledge about anammox cell biology. The anammox cell is divided into three separate compartments by bilayer membranes. The anammox cell consists of (from outside to inside) the cell wall, paryphoplasm, riboplasm, and anammoxosome. Not much is known about the composition or function of both the anammox cell wall and the paryphoplasm compartment. The cell wall is proposed to be proteinaceous and to lack both peptidoglycan and an outer membrane typical of Gram-negative bacteria. The function of the paryphoplasm is unknown, but it contains the cell division ring. The riboplasm resembles the standard cytoplasmic compartment of other bacteria; it contains ribosomes and the nucleoid. The anammoxosome occupies most of the cell volume and is a so-called "prokaryotic organelle" analogous to the eukaryotic mitochondrion. This is the site where the anammox reaction takes place, coupled over the curved anammoxosome membrane, possibly giving rise to a proton motive force and subsequent ATP synthesis. With these unique properties, anammox bacteria are food for thought concerning the early evolution of the domains Bacteria, Archaea, and Eukarya.

Correlation of macroscopic aggregation behavior and microscopic adhesion properties of bacteria strains using a dimensionless Tabor's parameter.

Macroscopic adhesion-aggregation, floc formation, and subsequent transportation of microorganisms in porous media are closely related to the microscopic behavior and properties of individual cells. The classical Tabor's parameter in colloidal science is modified to correlate the macroscopic aggregation and microscopic adhesion properties of microorganisms. Seven bacterial strains relevant to wastewater treatment and bioremediation were characterized in terms of their macroscopic aggregation index (AI) using an optical method, and their microscopic coupled adhesion and deformation properties using atomic force microscopy (AFM). Single cells were indented to measure the range and magnitude of the repulsive-attractive intersurface forces, elastic modulus, thickness and density of the cellular surface substances (CSS). The strong correlation suggests that cost and time effective microscopic AFM characterization is capable of making reliable prediction of macroscopic behavior.

The ultrastructure of the compartmentalized anaerobic ammonium-oxidizing bacteria is linked to their energy metabolism.

The most striking example of a complex prokaryotic intracytoplasmic organization can be found in the members of the phylum Planctomycetes. Among them are the anammox (anaerobic ammonium-oxidizing) bacteria, which possess a unique cell compartment with an unprecedented function in bacteria: the anammoxosome is a prokaryotic cell organelle evolved for energy metabolism. It is an independent entity, which is enclosed by a contiguous membrane. Several lines of evidence indicate its importance in the anammox reaction and the unusual subcellular organization may well be essential for the lifestyle of anammox bacteria. The present review summarizes our knowledge about the ultrastructure of anammox cells and the connection between the anammoxosome and the energy metabolism of the cell. In the future, much more research will be necessary to validate the current models and to answer questions on the functional cell biology of anammox bacteria.

Application of low intensity ultrasound to enhance the activity of anammox microbial consortium for nitrogen removal.

In this study, effect of low intensity ultrasound on the activity of anammox microbial consortium for nitrogen removal was investigated through batch experiments at the same irradiation frequency of 25 kHz. Total nitrogen removal rate increased by about 25.5% when ultrasound intensity of 0.3 w cm(-2) was applied at an optimal irradiation time of 4 min, and further experiments demonstrated that this effect could last for about 6 days. Analysis of extracellular polymeric substances indicated that the maximum increase of carbohydrate, protein and total extracellular substances was obtained on the first day after ultrasound, which was 28.8%, 30.5% and 29.7%, respectively. As the time prolonged, the production rate of extracellular carbohydrate, protein decreased gradually. Transmission electron microscopy observation demonstrated that ultrasounded cell wall of anammox microbial consortium became thinner resulting in increased release of extracellular substances. The results suggested that application of low intensity ultrasound may enhance the activity of anammox microbial consortium and ultimately the potential for nitrogen removal.

Morphology of bacterial granules developed in an upflow anaerobic acid reactor.

Macro- and micro-structures of granules, developed in an upflow anaerobic acid reactor, were examined by light and electron microscopy. Every granule was found to be white, soft and non-spherical and had an open cavity at the centre. Scanning electron microscopy showed that the granules were composed of rod-shaped bacteria, of different thicknesses and lengths, arranged in three distinct layers within the granules. Transmission electron microscopic analysis of ultra-thin sections of granules, stained with Ruthenium Red, revealed the presence of extra-cellular polymeric materials around the cells. Gram staining tests confirmed the presence of both Gram-positive and Gram-negative bacteria in the granules. The intertwined nature of the bacterial arrangement in the granules and the extracellular polymeric substance that encapsulated the cell colonies contributed to the structural stability of the granules.

Modeling effects of granules on the start-up of anaerobic digestion of dairy wastewater with Langmuir and extended Freundlich equations.

The effects of granules-inocula on the start-up of anaerobic reactors treating dairy manure were studied in a batch-fed reactor. The effects of start-up period and ratio of granules to feed were analyzed. Results indicated that the effects of start-up period could be described by Langmuir model, while the Extended Freundlich model could be used to model the effects of ratio of granules to feed on cumulative biogas production. In addition, transmission electron microscopes (TEM) and scanning electron microscope analysis were conducted to elucidate the distribution of microbial population and micro-colonies in granules and manure. From the TEM micrographs analyses, the ratios the Syntrophobacter and methanogens in granule and manure were shown to be 1.57 +/- 0.42 and 0.22 +/- 0.20, respectively. These results demonstrated that granules-inocula could reduce the period required for onset of biogas by 25%.

Testing various food-industry wastes for electricity production in microbial fuel cell.

Three food-industry wastes: fermented apple juice (FAJ), wine lees and yogurt waste (YW) were evaluated in combination with two sources of inoculum, anaerobic sludge and garden compost, to produce electricity in microbial fuel cells. Preliminary potentiostatic studies suggested that YW was the best candidate, able to provide up to 250 mA/m(2) at poised potential +0.3V/SCE. Experiments conducted with two-chamber MFCs confirmed that wine lees were definitely not suitable. FAJ was not able to start an MFC by means of its endogenous microflora, while YW was. Both FAJ and YW were suitable fuels when anaerobic sludge or compost leachate was used as inoculum source. Sludge-MFCs had better performance using YW (54 mW/m(2) at 232 mA/m(2)). In contrast, compost-leachate MFCs showed higher power density with FAJ (78 mW/m(2) at 209 mA/m(2)) than with YW (37 mW/m(2) at 144 mA/m(2)) but YW gave more stable production. Under optimized operating conditions, compost-leachate MFCs fueled with YW gave up to 92 mW/m(2) at 404 mA/m(2) and 44 mW/m(2) in stable conditions.

Decomposition and mineralization of aquatic humic substances (AHS) in treating landfill leachate using the Anammox process.

Aquatic humic substance (AHS) in landfill leachate is resistant to biodegradation, especially in anaerobic habitats. However, we reported here for the first time that AHS was completely biodegraded with the anaerobic ammonium oxidation (Anammox) process. As a result of aromatic-ring cleavage and mineralization, carboxylic and aliphatic organics were significantly produced, the contents of chromophores such as quinoid and ketone were remarkably decreased to decolorize the leachate, and produced carbon dioxide along with increasing carbonate was clearly presented. Of the degraded AHS of 137 mg L(-1), 51 mg L(-1) was owed to the oxidation with sulfate of 76 mg L(-1) as electron acceptor, and the rest to other metabolic mechanism. Isolation and identification of heterotrophic bacteria revealed a diversified consortium comprising four facultative anaerobic species, Bacillus sp., Paenibacillus sp., Bacteroides sp. and Staphylococcus sp., without sulfate-reducing bacteria detected. Their contribution to AHS biodegradation and sulfate reduction under the special conditions with high oxidization-reduction potential and insufficient electron acceptors has not been known yet. Further work is underway to investigate their properties and respective duties in the consortium.

Anti-infectious effect of S-benzylisothiourea compound A22, which inhibits the actin-like protein, MreB, in Shigella flexneri.

S-Benzylisothiourea compound A22 induces coccoid forms in Escherichia coli by inhibiting the function of the actin-like cytoskeletal protein, MreB. The minimum inhibitory concentration of A22 and the minimum concentration to induce coccoid forms for various pathogenic bacteria were determined. At 10 microg/ml, A22 induced coccoid forms in Shigella flexneri but did not inhibit the growth. No alteration of coccoid forms in the Gram-positive bacteria and anaerobic bacteria tested were observed following treatment with A22. To study the relationship between pathogenicity and alterations in bacterial shape, the infectious capacity of A22-induced coccoid S. flexneri was examined using CHO-K1 cells. Invasion of the coccoid cells was significantly reduced, however, no changes in adherence were observed. Using a mutant defective in the type III secretion apparatus, which delivers effectors to the host, we examined the secretion of effectors by A22-induced coccoid S. flexneri. The amount of secreted effectors in the coccoid cells was clearly decreased compared to rod-shaped cells. These results showed that the maintenance of rod-shaped cells by MreB in bacteria was essential for the secretion of effectors via the type III secretion system. Therefore, our results suggest that A22 is a useful lead compound for a novel anti-infectious agent without bactericidal activity and MreB is a candidate target site for development of new anti-infectious agents.