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Uropathogenic E. coli (UPEC) is the leading cause of urinary tract infections (UTI). These bacteria undertake a multi-stage infection cycle involving invasion of and proliferation within urinary tract epithelial cells, leading to the rupture of the host cell and dispersal of the bacteria, some of which have a highly filamentous morphology. Here we established a microfluidics-based model of UPEC infection of immortalized human bladder epithelial cells that recapitulates the main stages of bacterial morphological changes during the acute infection cycle in vivo and allows the development and fate of individual cells to be monitored in real-time by fluorescence microscopy. The UPEC-infected bladder cells remained alive and mobile in non-confluent monolayers during the development of intracellular bacterial communities (IBCs). Switching from a flow of growth medium to human urine resulted in immobilization of both uninfected and infected bladder cells. IBCs continued to develop and then released many highly filamentous bacteria via an extrusion-like process, whereas others showed strong UPEC proliferation yet no detected filamentation. The filamentation response was dependent on the weak acidity of human urine and required component(s) in a low molecular-mass (

Segmented filamentous bacteria (SFB) are known modulators of the mammalian immune system. Currently, the technology for investigating SFB culture in vitro is immature, and as a result, the mechanisms of SFB colonization and immune regulation are not yet fully elucidated. In this study, we investigated the gene diversity and host specificity of SFB flagellin genes. The fliC1 and fliC2 genes are relatively conserved, while the fliC3 and fliC4 genes are more variable, especially at the central and C-terminal regions. Host specificity analysis demonstrated that the fliC1 genes do not cluster together based on the host organism, whereas the fliC3 and fliC4 genes were host specific at the nucleotide and deduced amino acid levels. SFB flagellin protein expression in the ileum mucosa and cecal contents was detected by using fluorescence in situ hybridization (FISH) combined with immunohistochemical (IHC) analysis, immunoblotting, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Although the purified SFB FliC3 protein originating from both mouse and rat was able to activate Toll-like receptor 5 (TLR5)-linked NF-κB signaling, no host specificity was observed. Interestingly, the patterns of interaction with mouse ileum mucosal proteins were different for mouse FliC3 (mFliC3) and rat FliC3 (rFliC3). Gene Ontology (GO) and KEGG analyses indicated that more adherence-related proteins interacted with mFliC3, while more lysosome- and proteolysis-related proteins interacted with rFliC3. In vitro degradation experiments indicated that the stability of rFliC3 was lower than that of mFliC3 when they were incubated with mouse ileum mucosal proteins. In summary, the gene diversity and host specificity of SFB flagellin genes were investigated, and SFB flagellin expression was detected in gut samples. IMPORTANCE Since SFB genomes contain only one copy of each FliC gene, the diversity of FliC is representative of SFB strain diversity. Currently, little is known regarding the diversity and specificity of members of the group of SFB. The work presented herein demonstrates that select SFB strains, exhibiting unique FliC patterns, are present in a variety of mammalian hosts. SFB fliC genes were found to interact with a number of unique targets, providing further evidence for SFB host selection. Together, this work represents a major advancement in identifying SFB and delineating how members of the group of SFB interact with the host. Future examination of FliC genes will likely enhance our knowledge of intestinal colonization by the gut microbiota.

The cold-water coral Lophelia pertusa (Scleractinia, Caryophylliidae) is a key species in the formation of cold-water reefs, which are among the most diverse deep-sea ecosystems. It occurs in two color varieties: white and red. Bacterial communities associated with Lophelia have been investigated in recent years, but the role of the associated bacteria remains largely obscure. This study uses catalyzed reporter deposition fluorescence in situ hybridization to detect the in situ location of specific bacterial groups on coral specimens from the Trondheimsfjord (Norway). Two tissue-associated groups were identified: (i) bacteria on the host's tentacle ectoderm, “ Candidatus Mycoplasma corallicola,” are flasklike, pointed cells and (ii) endoderm-associated bona fide TM7 bacteria form long filaments in the gastral cavity. These tissue-bound bacteria were found in all coral specimens from the Trondheimsfjord, indicating a closer relationship with the coral compared to bacterial assemblages present in coral mucus and gastric fluid.

Th17 immunity in the gastrointestinal tract is regulated by the intestinal microbiota composition, particularly the presence of segmented filamentous bacteria (sfb), but the role of the intestinal microbiota in pulmonary host defense is not well explored. We tested whether altering the gut microbiota by acquiring sfb influences the susceptibility to staphylococcal pneumonia via induction of type 17 immunity. Groups of C57BL/6 mice which differed in their intestinal colonization with sfb were challenged with methicillin-resistant Staphylococcus aureus in an acute lung infection model. Bacterial burdens, bronchoalveolar lavage fluid (BALF) cell counts, cell types, and cytokine levels were compared between mice from different vendors, mice from both vendors after cohousing, mice given sfb orally prior to infection, and mice with and without exogenous interleukin-22 (IL-22) or anti-IL-22 antibodies. Mice lacking sfb developed more severe S. aureus pneumonia than mice colonized with sfb, as indicated by higher bacterial burdens in the lungs, lung inflammation, and mortality. This difference was reduced when sfb-negative mice acquired sfb in their gut microbiota through cohousing with sfb-positive mice or when given sfb orally. Levels of type 17 immune effectors in the lung were higher after infection in sfb-positive mice and increased in sfb-negative mice after acquisition of sfb, as demonstrated by higher levels of IL-22 and larger numbers of IL-22 + TCRβ + cells and neutrophils in BALF. Exogenous IL-22 protected mice from S. aureus pneumonia. The murine gut microbiota, particularly the presence of sfb, promotes pulmonary type 17 immunity and resistance to S. aureus pneumonia, and IL-22 protects against severe pulmonary staphylococcal infection.

Members of the monophyletic SOL cluster are large filamentous bacteria inhabiting the pelagic zone of many freshwater habitats. The abundances of SOL bacteria and compositions of SOL communities in samples from 115 freshwater ecosystems around the globe were determined by fluorescence in situ hybridization with cluster- and subcluster-specific oligonucleotide probes. The vast majority (73%) of sampled ecosystems harbored SOL bacteria, and all three previously described SOL subclusters (LD2, HAL, and GKS2-217) were detected. The morphometric and chemicophysical parameters and trophic statuses of ecosystems were related to the occurrence and subcluster-specific composition of SOL bacteria by multivariate statistical methods. SOL bacteria did not occur in acidic lakes (pH < 6), and their abundance was negatively related to high trophy and pH. The subcluster-specific variation in the compositions of SOL communities could be related to the pH, electrical conductivity, altitude, and trophic status of ecosystems. All three known SOL subclusters differed in respect to their tolerated ranges of pH and conductivity. Complete niche separation was observed between the vicarious subclusters GKS2-217 and LD2; the former occurred in soft-water lakes, whereas the latter was found in a broad range of hard-water habitats. The third subgroup (HAL) showed a wide environmental tolerance and was usually found sympatrically with the LD2 or GKS2-217 subcluster. Ecological differentiation of SOL bacteria at the subcluster level was most probably driven by differential adaptation to water chemistry. The distribution of the two vicarious taxa seems to be predominantly controlled by the geological backgrounds of the catchment areas of the habitats.

Filamentous bacteria with a conspicuous morphology were found in the majority of the bacterioplankton samples from a variety of freshwater habitats that were studied. These heterotrophic filaments typically account for Saprospiraceae . We developed a set of six nested probes for fluorescence in situ hybridization. Of the six probes, one probe was specific for Haliscomenobacter hydrossis , three probes were specific for the three subclusters (each probe was specific for one subcluster), one probe was specific for the entire SOL cluster, and another probe targeted almost the entire Saprospiraceae family. Specific hybridization of environmental samples and enrichments showed that the members of the three subclusters exhibited the same filamentous morphology. So far, using the subcluster-specific probes, we have not been able to detect any bacteria with a differing morphology. We conclude that the SOL cluster bacteria are an integral part of bacterioplankton in many freshwater habitats. They potentially account for a large fraction of the total bacterial biomass but have been underrepresented in molecular diversity studies so far.

Ephemeral blooms of filamentous bacteria are a common phenomenon in the water column of oligo- to mesotrophic lakes. It is assumed that the appearance of such morphotypes is favored by selective predation of bacterivorous protists and that filter-feeding zooplankton plays a major role in suppressing these bacteria. The phylogenetic affiliation of the important bloom-forming filamentous bacteria in freshwaters is presently unknown. Here we report the identification of dominant members of a filamentous bacterial assemblage during a bloom of such morphotypes in a mesotrophic lake. By molecular cloning and fluorescence in situ hybridization with specific oligonucleotide probes, up to 98% of filamentous cells in lake water could be assigned to a clade of almost identical (99% similarity) 16S rRNA gene sequence types, the cosmopolitan freshwater LD2 cluster. For a period of less than 1 week, members of the LD2 clade constituted >40% of the total bacterial biomass, potentially favored by high grazing of planktivorous protists. This is probably the most pronounced case of dominance by a single bacterioplankton species ever observed in natural freshwaters. In enclosures artificially stocked with the metazoan filter feederDaphnia, bacteria related to the LD2 clade formed a significantly larger fraction of filaments than in enclosures whereDaphniahad been removed. However, in the presence of higher numbers ofDaphniaindividuals, the LD2 bacteria, like other filaments, were eventually eliminated both in enclosures and in the lake. This points at the potential importance of filter-feeding zooplankton in controlling the occurrence and species composition of filamentous bacterial morphotypes in freshwater plankton.

Current models suggest that (i) filamentous bacteria are protected against predation by nanoflagellates, (ii) prey size is positively correlated with prey-predator contact probability, and (iii) contact probability is mainly responsible for size-selective predation by interception-feeding flagellates. We used five strains of filamentous bacteria and one bacterivorous nanoflagellate, Ochromonas sp. strain DS, to test these assumptions. The five strains, including one spirochete and four Betaproteobacteria strains, were isolated by the filtration-acclimatization method. All five strains possess flexible cells, but they differ in average cell length, which ranged from 4.5 to 13.7 μm. High-resolution video microscopy was used to measure contact, capture, and ingestion rates, as well as selectivity of the flagellate feeding. Growth and feeding experiments with satiating and nonsatiating food conditions, as well as experiments including alternative well-edible prey, were performed. In contrast to predictions by current models, the flagellate successfully consumed all the tested filamentous strains. The ingestion rate was negatively correlated with bacterial length. On the other hand, the lengths of the filamentous bacteria were not positively correlated to the contact rate and capture rate but were negatively correlated to ingestion efficiency. In experiments including alternative nonfilamentous prey, the flagellates showed negative selection for filamentous bacteria, which was independent of food concentration and is interpreted as a passive selection. Our observations indicate that (i) size alone is not sufficient to define a refuge for filamentous bacteria from nanoflagellate predation and (ii) for the investigated filamentous bacteria, prey-predator contact probability could be more influenced by factors other than the prey size.

A recently discovered bacterial/archaeal association, growing in a string-of-pearls-like structure, thrives in the cold (∼10°C) sulfidic marsh water of the Sippenauer Moor near Regensburg, Bavaria, Germany. It forms characteristic, macroscopically visible globules, the pearls, containing microcolonies of novel euryarchaeota, which are surrounded by mainly filamentous bacteria (C. Rudolph, G. Wanner, and R. Huber, Appl. Environ. Microbiol. 67:2336-2344, 2001). Single pearls in series are connected by white threads. Here we report the first detailed molecular investigations of the taxonomic affiliation of the bacteria contributing to the strings of pearls. Phylogenetic analysis showed the dominance of a single phylotype (clone sipK4) within single pearls most closely related to Thiothrix unzii . The presence of Thiothrix sipK4 as a major constituent of single pearls and of the pearl-connecting white threads was verified by fluorescence in situ hybridization.

Two filamentous bacteria causing bulking in two activated sludges were examined. Investigations using morphological features, staining techniques, and fluorescent in situ hybridization identified both filaments as type 021N. However, an examination of the effect of chlorine on the sludges revealed a chlorine-susceptible type 021N in one sludge and a chlorine-resistant type 021N in the other.

Segmented filamentous bacteria (SFB) are autochthonous bacteria colonizing the ileum of many young animals by attaching to intestinal epithelial cells. These nonpathogenic bacteria strongly stimulate the mucosal immune system and induce intestinal epithelial cells to express major histocompatibility complex class II molecules. We tried to discover whether SFB are phagocytized and intracellularly processed by the host cells, which is indicative of antigen processing. The middle part of the ileum was extracted from 10- and 20-day-old broiler chicks ( Gallus gallus domesticus ). Samples were processed and examined by scanning and transmission electron microscopy (SEM and TEM, respectively). In SEM, no, few, medium, and dense SFB colonization levels were classified. In TEM of cells from animals with medium or dense SFB colonization levels, we could observe extracellular particles ranging from those only indenting the cell membrane to particles found in the cytoplasmatic area beyond the terminal web. These particles had a structural similarity with SFB that were floating freely in the intestinal lumen. Furthermore, we observed unlacing of the membrane and septum surrounding the extracellular particles and their incorporation into host cytoplasmatic components, which strongly suggests that these particles are phagocytized and intracellularly processed SFB. This conclusion is supported by TEM analysis of samples with no or few SFB, in which we failed to find these characteristic morphologies. The phagocytosis process described here could be an important trigger for the stimulating effect of SFB on the mucosal immune system.

Four strains of filamentous bacteria were isolated from slimes collected in different paper mill factories. Morphological and physiological characterization of the isolates indicated an affiliation with the genus Sphaerotilus . However, while the physiological properties of the isolates were almost identical, pronounced physiological differences between the isolates and Sphaerotilus natans DSM 6575 T , DSM 565, and DSM 566 with respect to their ability to metabolize complex polysaccharides, sugars, polyalcohols, or organic acids as carbon sources were detected. In contrast to the analyzed culture collection strains of S. natans , all paper mill isolates were able to grow at elevated temperatures of up to 40°C. Comparative sequence analysis of nearly complete 16S ribosomal DNA (rDNA) sequences from the four new isolates demonstrated that the retrieved sequences were highly similar to each other (99.6 to 99.8% similarity) and to previously published partial 16S rDNA sequences of S. natans DSM 6575 T and ATCC 15291. Polyphasic characterization of the isolated Sphaerotilus strains revealed interesting adaptations of the strains to the environmental paper mill conditions with regard to temperature tolerance and utilization of cellulose and starch.

Uropathogenic Escherichia coli (UPEC) strains are capable of invading bladder epithelial cells (BECs) on the bladder luminal surface. Based primarily on studies in mouse models, invasion is proposed to trigger an intracellular uropathogenic cascade involving intracellular bacterial proliferation followed by escape of elongated, filamentous bacteria from colonized BECs. UPEC filaments on the mouse bladder epithelium are able to revert to rod-shaped bacteria, which are believed to invade neighboring cells to initiate new rounds of intracellular colonization. So far, however, these late-stage infection events have not been replicated in vitro . We have established an in vitro model of human bladder cell infection by the use of a flow chamber (FC)-based culture system, which allows investigation of steps subsequent to initial invasion. Short-term bacterial colonization on the FC-BEC layer led to intracellular colonization. Exposing invaded BECs to a flow of urine, i.e., establishing conditions similar to those faced by UPEC reemerging on the bladder luminal surface, led to outgrowth of filamentous bacteria similar to what has been reported to occur in mice. These filaments were capable of reverting to rods that could invade other BECs. Hence, under growth conditions established to resemble those present in vivo , the elements of the proposed uropathogenic cascade were inducible in a human BEC model system. Here, we describe the model and show how these characteristics are reproduced in vitro .

Segmented filamentous bacteria (SFBs) are apathogenic autochthonous bacteria in the murine small intestine that preferentially attach to Peyer's patch epithelium. SFBs have never been cultured in vitro. We have studied the effects of SFBs on the immune system of the host. Mice monoassociated with SFBs were compared with germ-free mice and with mice without SFBs but with a specific-pathogen-free (SPF) gut flora. SFBs versus no microbial flora raised the number of lymphoid cells in the lamina propria of the ileal and cecal mucosa, raised the number of immunoglobulin A (IgA)-secreting cells in the intestinal mucosa, produced elevated IgA titers in serum and intestinal secretions, and enhanced the concanavalin A-induced proliferative responses of mesenteric lymph node cells. The SPF flora had effects similar to but less pronounced than those mediated by SFBs. The results indicate that SFBs stimulate the mucosal immune system to a greater extent than do other autochthonous gut bacteria.

Filamentous bacteria in marine sediments can behave as “living electrical cables,” says microbial ecologist Lars Nielsen of Aarhus University in Aarhus, Denmark. Those cables extend across considerable distances in microbial terms, enabling these bacteria to conduct electric currents on a “centimeter scale.” He spoke during the symposium “Enviable Microbial Powers to Tap Earth's Energy,” convened as part of the 2012 ASM General Meeting in San Francisco last June.

Segmented filamentous bacteria (SFB) are found in multiple species and play an important role in the development of mucosal immunity. The mechanism by which the bacteria interact with the immune system has not been well defined. We provide morphologic evidence of direct interaction between SFB and intraepithelial mononuclear cells.

Fifteen filamentous strains, morphologically classified as Eikelboom type 021N bacteria, were isolated from bulking activated sludges. Based on comparative 16S ribosomal DNA (rDNA) sequence analysis, all strains form a monophyletic cluster together with all recognized Thiothrix species (88.3 to 98.7% 16S rDNA sequence similarity) within the gamma-subclass of Proteobacteria . The investigated Eikelboom type 021N isolates were subdivided into three distinct groups (I to III) demonstrating a previously unrecognized genetic diversity hidden behind the uniform morphology of the filaments. For in situ detection of these bacteria, 16S rRNA-targeted oligonucleotide probes specific for the entire Eikelboom type 021N- Thiothrix cluster and the Eikelboom type 021N groups I, II, and III, respectively, were designed, evaluated, and successfully applied in activated sludge.

Scanning electron microscopy, light microscopy, and direct isolations were used to examine the distribution and diversity of bacteria in the gut tracts of larval stages of Tipula abdominalis . The animal had an enlarged hindgut which housed a diverse bacterial community in the lumen and directly attached to the gut wall. Distinct localization was noted, with the most dense and most diverse community anterior to the rectum. A distinct architecture of bacteria occurred in this region, characterized by a layering or a “weblike” array of filamentous bacteria overlying mats of bacteria closely associated with the gut wall. Although morphological diversity was high in the hindgut, filamentous bacteria were the dominant morphology observed. The attached microbiota, sloughed during ecdysis, recolonized to the same density and diversity observed before the molt. The majority of the isolatable bacterial types were facultatively anaerobic. The distinct localization and attached nature of the hindgut bacteria and the recolonization after each molt suggest they are indigenous to this region of the gut tract. Images

Recently, it was suggested that a mannose-specific lectin on the bacterial cell surface is responsible for the recognition by phagocytic cells of certain nonopsonized Escherichia coli strains. In this study we assessed the interaction of two strains of E. coli at different phases of growth with a monolayer of mouse peritoneal macrophages and developed a direct method with [ 14 C]mannan to quantitate the bacterial mannose-binding activity. Normal-sized bacteria were obtained from logarithmic and stationary phases of growth. Nonseptated filamentous cells were formed by growing the organisms in the presence of cephalexin or at a restrictive temperature. Attachment to macrophages of all bacterial forms was inhibited by methyl α- d -mannoside and mannan but not by other sugars tested. The attachment of stationary phase and filamentous bacteria to macrophages, as well as their mannose-binding activity, was similar, whereas in the exponential-phase bacteria they were markedly reduced. The results show a linear relation between the two parameters ( R = 0.98, P < 0.001). The internalization of the filamentous cells attached to macrophages during 45 min of incubation was much less efficient (20%) compared to that of exponential-phase, stationary-phase, or antibody-coated filamentous bacteria (90%). The results indicate that the mannose-binding activity of E. coli determines the recognition of the organisms by phagocytes. They further suggest that administration of β-lactam antibiotics may impair elimination of certain pathogenic bacteria by inducing the formation of filaments which are inefficiently internalized by the host's phagocytic cells.

Sphaerotilus natans , one of the most widely reported causes of bulking in activated sludge, can exist both within and outside of a sheath. It can easily be confused with similar activated sludge bacteria and thus can be overlooked when present in low numbers. Fluorescent antiserum was successfully prepared against the nonfilamentous form and was shown to be highly specific, showing no reaction with either pure cultures of similar filamentous bacteria or entirely unrelated organisms. It did, however, show a lack of strain specificity since it reacted with S. natans isolates from the Federal Republic of Germany and the United States and with filamentous bacteria in South African activated sludges. Fluorescent antibody is capable of penetrating the filaments of S. natans to stain the cells individually. The use of fluorescent antiserum in the identification of S. natans filaments obscured by activated sludge flocs and other suspended matter was simple since the cells stained brightly and could be observed through the less dense matter, while the use of other microscope techniques would be hampered by these obstructions. The use of fluorescent antibody will facilitate ecological studies of S. natans in activated sludge and other aqueous environments.

A method was developed to assess the activity of filamentous bacteria in activated sludge. It involves the incubation of activated sludge with 2( p -iodophenyl)-3-( p -nitrophenyl)-5-phenyl tetrazolium chloride followed by staining with malachite green. Both cells and 2( p -iodophenyl)-3-( p -nitrophenyl)-5-phenyl tetrazolium chloride-formazan crystals can be observed in prepared specimens by using bright-field microscopy. This procedure allowed us to distinguish between inactive and actively metabolizing filaments after chlorine application to control the bulking of activated sludge.

Polyclonal antiserum prepared against Chloroflexus aurantiacus reacted with all Chloroflexus strains examined but not with other morphologically or physiologically similar bacteria. Only one of three filament types in a natural hot spring cyanobacterial mat reacted with this antiserum. Reacting filaments remained antigenically positive deep within the mat in material estimated to be several years old.