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.

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.

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.

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.

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.

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.

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.

Detoxification of vinyl chloride to ethene coupled to growth of an anaerobic bacterium.

Tetrachloroethene (PCE) and trichloroethene (TCE) are ideal solvents for numerous applications, and their widespread use makes them prominent groundwater pollutants. Even more troubling, natural biotic and abiotic processes acting on these solvents lead to the accumulation of toxic intermediates (such as dichloroethenes) and carcinogenic intermediates (such as vinyl chloride). Vinyl chloride was found in at least 496 of the 1,430 National Priorities List sites identified by the US Environmental Protection Agency, and its precursors PCE and TCE are present in at least 771 and 852 of these sites, respectively. Here we describe an unusual, strictly anaerobic bacterium that destroys dichloroethenes and vinyl chloride as part of its energy metabolism, generating environmentally benign products (biomass, ethene and inorganic chloride). This organism might be useful for cleaning contaminated subsurface environments and restoring drinking-water reservoirs.

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%.

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.

Enrichment and characterization of marine anammox bacteria associated with global nitrogen gas production.

Microbiological investigation of anaerobic ammonium oxidizing (anammox) bacteria has until now been restricted to wastewater species. The present study describes the enrichment and characterization of two marine Scalindua species, the anammox genus that dominates almost all natural habitats investigated so far. The species were enriched from a marine sediment in the Gullmar Fjord (Sweden) using a medium based on Red Sea salt. Anammox cells comprised about 90% of the enrichment culture after 10 months. The enriched Scalindua bacteria displayed all typical features known for anammox bacteria, including turnover of hydrazine, the presence of ladderane lipids, and a compartmentalized cellular ultrastructure. The Scalindua species also showed a nitrate-dependent use of formate, acetate and propionate, and performed a formate-dependent reduction of nitrate, Fe(III) and Mn(IV). This versatile metabolism may be the basis for the global distribution and substantial contribution of the marine Scalindua anammox bacteria to the nitrogen loss from oxygen-limited marine ecosystems.

Candidatus "Anammoxoglobus propionicus" a new propionate oxidizing species of anaerobic ammonium oxidizing bacteria.

The bacteria that mediate the anaerobic oxidation of ammonium (anammox) are detected worldwide in natural and man-made ecosystems, and contribute up to 50% to the loss of inorganic nitrogen in the oceans. Two different anammox species rarely live in a single habitat, suggesting that each species has a defined but yet unknown niche. Here we describe a new anaerobic ammonium oxidizing bacterium with a defined niche: the co-oxidation of propionate and ammonium. The new anammox species was enriched in a laboratory scale bioreactor in the presence of ammonium and propionate. Interestingly, this particular anammox species could out-compete other anammox bacteria and heterotrophic denitrifiers for the oxidation of propionate in the presence of ammonium, nitrite and nitrate. We provisionally named the new species Candidatus "Anammoxoglobus propionicus".

Biohydrogen production from chemical wastewater treatment in biofilm configured reactor operated in periodic discontinuous batch mode by selectively enriched anaerobic mixed consortia.

Molecular hydrogen (H(2)) production with simultaneous wastewater treatment was studied in biofilm configured periodic discontinuous/sequencing batch reactor using chemical wastewater as substrate. Anaerobic mixed consortia was sequentially pretreated with repeated heat-shock (100 degrees C; 2 h) and acid (pH-3.0; 24 h) treatment procedures to selectively enrich the H(2) producing mixed consortia prior to inoculation of the reactor. The bioreactor was operated at mesophilic (room) temperature (28+/-2 degrees C) under acidophilic conditions with a total cycle period of 24 h consisting of FILL (15 min), REACT (23 h), SETTLE (30 min) and DECANT (15 min) phases. Reactor was initially operated with synthetic wastewater (SW) at OLR of 4.8 kg COD/m(3)-day and subsequently operated using composite chemical wastewater (CW) at OLR of 5.6 kg COD/m(3)-day by adjusting pH to 6.0 prior to feeding to inhibit the methanogenic activity. H(2) evolution rate differed significantly with the nature of wastewater used as substrate [SW--volumetric H(2) production rate--12.89 mmol H(2)/m(3)-min and specific H(2) production rate--0.0084 mmol H(2)/min-g COD(L) (0.026 mmol H(2)/min-g COD(R)); CW--volumetric H(2) production rate--6.076 mmol H(2)/m(3)-min and specific H(2) production rate--0.0089 mmol H(2)/min-g COD(L) (0.033 mmol H(2)/min-g COD(R))]. Relatively rapid progress towards higher H(2) yield (2 h) was observed with SW compared to the CW (10 h). Substrate (COD) reduction of 32.4% (substrate degradation rate (SDR)--1.55 kg COD/m(3)-day) and 26.7% (SDR-1.49 kg COD/m(3)-day) was observed with SW and CW, respectively. The system showed rapid stabilization tendency (SW--37 days; CW--40 days) with respect to H(2) generation and COD reduction. H(2) evolution showed relatively good correlation with VFA concentration in the case of SW (R(2)-0.961) compared to CW (R(2)-0.912). A surge in pH values from 5.87 to 4.23 (SW) and 5.93 to 4.62 (CW) was observed during the cycle operation. Integration of biofilm configuration with periodic discontinuous batch operation under the defined operating conditions showed potential to influence the microbial system by selectively enriching the specific group of microflora capable of producing H(2).

The performance of a phase separated granular bed bioreactor treating brewery wastewater.

This study presents the performance characteristics of a plug flow phase separated anaerobic granular bed baffled reactor (GRABBR) fed with brewery wastewater at various operating conditions. The reactor achieved chemical oxygen demand (COD) removal of 93-96% with high methane production when operated at organic loading rates (OLRs) of 2.16-13.38kg COD m(-3)d(-1). The reactor configuration and microbial environment encouraged the acidogenic dominant zone to produce intermediate products suitable for degradation in the predominantly methanogenic zone. Noticeable phase separation between acidogenesis and methanogenesis mainly occurred at high OLR, involving a greater number of compartments to contribute to wastewater treatment. The highly active nature and good settling characteristics of methanogenic granular sludge offered high biomass retention and enhanced methanogenic activities within the system. The granular structure in the acidogenic dominant zone of the GRABBR was susceptible to disintegration and flotation. Methanogenic granular sludge was a multi-layered structure with Methanosaeta-like organisms dominant in the core.

Effect of limited aeration on the anaerobic treatment of evaporator condensate from a sulfite pulp mill.

Serious inhibition was found in the regular up-flow anaerobic sludge blanket (UASB) reactor in treating the evaporator condensate from a sulfite pulp mill, which contained high strength sulfur compounds. After applying the direct limited aeration in the UASB, the inhibition was alleviated gradually and the activity of the microorganisms was recovered. The COD removal rate increased from 40% to 80% at the organic loading rate of 8kgCODm(-3)d(-1) and a hydraulic retention time of 12h. Limited aeration caused no oxygen inhibition to the anaerobic microorganisms but instigated sulfide oxidization and H(2)S removal, which was beneficial to the methanogens. The experiment confirmed the feasibility of applying limited aeration in the anaerobic reactor to alleviate the sulfide inhibition. It also proved that the anaerobic system was actually aerotolerant. SEM observation showed that the predominant microorganisms partly changed from rod-shaped methanogens to cocci after the UASB reactor was aerated.

Anammox enrichment from different conventional sludges.

Three sets of sequencing batch reactor (SBR) were used for Anammox enrichment from conventional sludges including upflow anaerobic sludge blanket, activated sludge, and anaerobic digestion sludge. After four months of operation, the Anammox activity occurred in all reactors allowing continuous removal of ammonium and nitrite. The morphology of the cultivated Anammox sludge was observed using scanning electron microscope. The photographs showed that the obtained culture was mostly spherical in shape, presumably Anammox culture. There were also filamentous-like bacteria co-existing in the system. Fluorescence in situ hybridization (FISH) analysis using 16S rRNA targeting oligonucleotide probes PLA46 and Amx820 showed that the dominant population developed in all SBRs was hybridized with both PLA46 and Amx820 gene probes. It means that the cultivated biomass in all SBRs was classified in the group of Planctomycetales bacteria with respect to the anaerobic ammonium-oxidizing bacteria, Candidatus Brocadia anammoxidans and Candidatus Kuenenia stuttgartiensis. Numerous time sequences were tested in this experiment. The shortest workable reaction time was found in the range from 5 to 7 h. Good quiescence of sludge was obtained at 30 min of settle period followed by a discharge period of 15 min. A long-term performance showed a near perfect removal of nitrite based on the influent NO2(-)-N concentration of 50-70 mg l(-1). The maximum ammonia removal efficiency was 80% with the influent NH4(+)-N concentration of 40-60 mg l(-1). It is, therefore, concluded that Anammox cultivation from conventional sludges was highly possible under control environment within four months.

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.

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.

Enrichment and isolation of anaerobic hydrocarbon-degrading bacteria.

Recent progress in microbiology resulted in the enrichment and isolation of anaerobic bacteria capable of the biodegradation of various hydrocarbons under a variety of electron-accepting conditions. Problems challenging the enrichment and isolation of anaerobic hydrocarbonclastic organisms required new approaches and modifications of conventional microbiological techniques. This chapter summarizes the collective experience accumulated in this area starting from anaerobic sampling precautions and includes all stages of cultivation from the construction of initial incubations to final isolation steps and the evaluation of culture purity.