Eubacterial Probe Research Articles

Fluorescent In Situ Hybridization for the Detection of Intracellular Bacteria in Companion Animals.

FISH techniques have been applied for the visualization and identification of intracellular bacteria in companion animal species. Most frequently, these techniques have focused on the identification of adhesive-invasive Escherichia coli in gastrointestinal disease, although various other organisms have been identified in inflammatory or neoplastic gastrointestinal disease. Previous studies have investigated a potential role of Helicobacter spp. in inflammatory gastrointestinal and hepatic conditions. Other studies evaluating the role of infectious organisms in hepatopathies have received some attention with mixed results. FISH techniques using both eubacterial and species-specific probes have been applied in inflammatory cardiovascular, urinary, and cutaneous diseases to screen for intracellular bacteria. This review summarizes the results of these studies.

Bacterial Biogeography of the Colon in Dogs With Chronic Inflammatory Enteropathy.

The intestinal microbiota is believed to play a role in the pathogenesis of inflammatory bowel disease in humans and chronic inflammatory enteropathy (CIE) in dogs. While most previous studies have described the gut microbiota using sequencing methods, it is fundamental to assess the spatial distribution of the bacteria for a better understanding of their relationship with the host. The microbiota in the colonic mucosa of 22 dogs with CIE and 11 control dogs was investigated using fluorescence in situ hybridization (FISH) with a universal eubacterial probe (EUB338) and specific probes for select bacterial groups. The number of total bacteria labeled with EUB338 probe was lower within the colonic crypts of dogs with CIE compared to controls. Helicobacter spp. and Akkermansia spp. were decreased on the colonic surface and in the crypts of dogs with CIE. Dogs with CIE had increased number of Escherichia coli/Shigella spp. on the colonic surface and within the crypts compared to control dogs. In conclusion, the bacterial microbiota in the colonic mucosa differed between dogs with and without CIE, with depletion of the crypt bacteria in dogs with CIE. The crypt bacterial species that was intimately associated with the host mucosa in control dogs was composed mainly of Helicobacter spp.

Identification of Mucosa-Invading and Intravascular Bacteria in Feline Small Intestinal Lymphoma

Persistent bacterial infections of the gastrointestinal mucosa are causally linked to gastric carcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma in people and laboratory animals. We examined the relationship of mucosa-associated bacteria to alimentary lymphoma in cats. Intestinal biopsies from 50 cats with alimentary lymphoma (small cell, n = 33; large cell, n = 17) and 38 controls without lymphoma (normal to minimal change on histopathology, n = 18; lymphocytic-plasmacytic enteritis, n = 20) were evaluated. The number and spatial distribution of bacteria (ie, in luminal cellular debris, villus-associated mucus, adherent to epithelium, mucosal invasion, intravascular, or serosal) were determined by fluorescence in situ hybridization with the eubacterial probe EUB-338. Mucosa-invasive bacteria were more frequently observed in cats with large cell lymphoma (82%, P ≤ .001) than in cats with small cell lymphoma (18%), normal to minimal change on histopathology, and lymphocytic-plasmacytic enteritis (3%). Intravascular bacteria were observed solely in large cell lymphoma (29%), and serosal colonization was more common in cats with large cell lymphoma (57%) than with small cell lymphoma (11%, P ≤ .01), normal to minimal change (8%, P ≤ .01), and lymphocytic-plasmacytic enteritis (6%, P ≤ .001). The high frequency of invasive bacteria within blood vessels and serosa of cats with large cell lymphoma may account for the sepsis-related complications associated with large cell lymphoma and inform clinical management. Further studies are required to determine the role of intramucosal bacteria in the etiopathogenesis of feline alimentary lymphoma.

Goblet cell Hypoxia-Inducible Factor (HIF) is necessary for mucin processing and maintenance of mucosal integrity

Abstract Secreted mucus is essential for intestinal homeostasis, separating luminal bacteria from the colonic epithelium and preventing inappropriate inflammation. Perturbations in goblet cell mucus secretion have been shown to lead to spontaneous colitis in mice. Treatment of mice with a prolyl hydroxylase inhibitor (AKB-4924) leads to stabilization of hypoxia inducible factor-1 alpha (HIF-1α) and regulation of hypoxia-responsive genes that promote mucosal protection. Interestingly, goblet cell hyperplasia was observed following in vivo administration of AKB-4924, which may account for the mucosal protection observed. Thus, we hypothesized epithelial stabilization of HIF-1α leads to goblet cell differentiation. Utilizing 3D murine colonic organoid system, treated with a gamma secretase inhibitor (DBZ), AKB-4924 or hypoxia, we analyzed changes in differentiation markers by qPCR. Contrary to our hypothesis, neither AKB-4924 nor hypoxia resulted in the induction of goblet cell differentiation factors. Furthermore, mice lacking HIF-1α in the intestinal epithelium (HIF-1αfl/fl-VillinCRE) exhibited normal colonic goblet cell numbers. However, these goblet cells appeared less organized with diffuse mucin granules. Fluorescent in situ hybridization with a eubacterial probe revealed diminished thickness, with reduced proximity to the microbiome in HIF−/− mice. Finally, profiling the composition of the mucus layer revealed significantly altered expression profile of both secreted and adherent mucins. We conclude that epithelial HIF-1α alters goblet cell function, regulating intestinal mucus, which may contribute to the mucosal-protective role of HIF.

English

Activated sludge samples were used to analyse microbial community structures from the anoxic and aerobic zones of a labora­tory-scale modified Ludzack-Ettinger system. Fluorescent in situ hybridization and denaturing gradient gel electrophoresis were applied for analysis. With the help of DNA specific fluorochrome DAPI, approximately 75 to 80% of total cells were detected, hybridised with a specific eubacterial probe for the anoxic and aerobic zones. Results corroborate the dominance of the alpha and gamma subclasses of the Proteobacteria in the anoxic zone whilst the aerobic zone was dominated with the beta subclass of the Proteobacteria. Genetic diversity of the microbial community present in each of the anoxic and aerobic zones was employed by the DGGE technique. Results were obtained from the application of fluorescent in situ hybridisation (FISH) and PCR-DGGE yields a more precise understanding of the microbial community structure and genetic diversity present in domestic wastewater of a laboratory scale treatment process. Nitrogen mass balances indicated an upset in the nitrogen levels for wastewater batches two and seven. The carbon mass balance fell in the range of 92.4 and 105.9% and the nitrogen mass balance fell in the range of 98.4 and 160.0%. Key words: Fluorescent in situ hybridisation (FISH), denaturing gradient gel electrophoresis (DGGE), COD and nitrogen mass balances.

Evaluation of the diagnostic value of fluorescent in situ hybridization in a rat model of bacterial pneumonia

In severe nosocomial pneumonia, the pathogenic responsibility of bacteria isolated from airways is far from certain, and a lung biopsy is sometimes performed. However, detection and identification of pathogens are frequently unachieved. Here, we developed a protocol for direct visualization of bacteria within the lung tissue using fluorescent in situ hybridization (FISH) in a rat model of Acinetobacter baumannii pneumonia. The reference positive diagnosis of bacterial pneumonia was the presence of pathological signs of pneumonia associated with the proof of bacteria or bacterial PCR products into the parenchyma. By analysis of 122 sets of slices from 26 rats and using the eubacterial probe EUB-338, our results show that FISH reached a sensitivity and a diagnostic accuracy higher than that of optic microscopy (sensitivity: 96% versus 55.4% and diagnostic accuracy: 96.7% versus 66.4%), whereas both approaches had 100% specificity. FISH could be useful especially on negative biopsies from patients with suspected infectious pneumonia.

674 Confocal Laser Endomicroscopy for In Vivo Diagnosis of Clostridium Difficile Associated Colitis

Confocal Laser Endomicroscopy for In Vivo Diagnosis of Clostridium Difficile Associated Colitis Helmut Neumann, Michael Vieth, Martin Grauer, Raja Atreya, Jonas Mudter, Christoph Becker, Nadine Wittkopf, Markus F. Neurath Department of Medicine 1, University of Erlangen-Nuremberg, Erlangen, Germany; Institute of Pathology, Klinikum Bayreuth, Bayreuth, Germany Background: Clostridium difficile infection (CDI) is one of the most clinically significant causes of hospital-acquired diarrhea and associated with significant morbidity and mortality. Endomicroscopy (CLE) has recently risen as a new emerging endoscopic imaging modality enabling real time in vivo histology during ongoing endoscopy. Aims: Main objective was to investigate whether CLE has the capability for the in vivo diagnosis of CDI. Second objective of our study was to proof the presence of intramucosal bacteria using CLE. Material & Methods: Prospectively, seventy-eight consecutive patients with diarrhea were included of whom eight patients were diagnosed with CDI based on toxigenic culture. CLE was performed after intravenous injection of fluorescein. Additionally, in three patients topical application of acriflavine hydrochloride was performed. In order to validate the presence of intramucosal bacteria ex vivo CLE was performed in pure C. difficile culture; in addition fluorescence in situ hybridization (FISH) was made. Finally, endomicroscopy with fluorescence labelled eubacterial oligonucleotide probes specific for C. difficile was performed in order to prove the presence of intramucosal bacteria. Results: Overall, 80 lesions were examined using endomicroscopy. CLE could readily identify CDIassociated histological changes in vivo. In early CDI, CLE revealed small surface erosions of the superficial colonic crypts and an increased cellular infiltrate. Microvessels within the lamina propria were slightly dilated but showed no leakage. In advanced CDI, CLE demonstrated massively dense cellular infiltrate. Normal colonic appearance was nearly completely abolished and fragile vessels with leakage became visible. Furthermore, a plaque of loosely cells, fibrin and debris covered the mucosal surface. Sensitivity and specificity of CLE in predicting CDI was 75% and 92.9%, respectively. In addition, intramucosal bacteria were visualized using CLE. The presence of these bacteria could also be proven by CLE with fluorescein-labelled specific C. difficile probes and by using the FISH-technique. Sensitivity and specificity for the presence of intramucosal bacteria on endomicroscopy and afterwards confirmed with FISH was 100%. No adverse events regarding the procedure or the use of fluorescence agents were observed. Conclusions: CLE has the potential for the in vivo diagnosis of CDI associated colitis thus enabling accelerated diagnosis and improved patient management. In addition, CLE enabled the detection of intramucosal bacteria in vivo which could open a plethora of new indications in the near future.

The continuing search for Mycobacterium tuberculosis involvement in sarcoidosis: a study on archival biopsy specimens

Increasing evidence indicates that mycobacteria may be involved in the aetiology and pathophysiology of sarcoidosis. To investigate the association between Mycobacterium tuberculosis complex infection and sarcoidosis. Mediastinal lymph node biopsy specimens (formalin-fixed, paraffin-embedded) from 52 Danish patients with sarcoidosis, 50 patients with mediastinal lymphadenopathy of other non-mycobacterial causes (negative controls) and 12 patients with histologically and/or culture-verified mycobacteriosis (positive controls) were included in the study. Biopsy samples were analysed for the presence of Mycobacterium tuberculosis complex by strand displacement assay and a subset of specimens were examined for bacterial rRNA by fluorescent in situ hybridisation using an eubacterial probe with general bacterial specificity (EUB338). One patient with sarcoidosis displayed a positive M. tuberculosis complex test. All negative controls were negative in the test and 5/12 patients with mycobacteriosis were positive in the test. We detected M. tuberculosis complex DNA in 10-year-old biopsy samples. Thirty-six samples were tested with the eubacterial probe; of these, 67% were positive with no difference between patients and controls. Our results do not support the hypothesis that M. tuberculosis complex infection is involved in the pathogenesis of sarcoidosis. However, we stress the importance of excluding mycobacteriosis in the diagnostic workup of sarcoidosis patients.

Bacterial acquisition in juveniles of several broadcast spawning coral species.

Coral animals harbor diverse microorganisms in their tissues, including archaea, bacteria, viruses, and zooxanthellae. The extent to which coral-bacterial associations are specific and the mechanisms for their maintenance across generations in the environment are unknown. The high diversity of bacteria in adult coral colonies has made it challenging to identify species-specific patterns. Localization of bacteria in gametes and larvae of corals presents an opportunity for determining when bacterial-coral associations are initiated and whether they are dynamic throughout early development. This study focuses on the early onset of bacterial associations in the mass spawning corals Montastraea annularis, M. franksi, M. faveolata, Acropora palmata, A. cervicornis, Diploria strigosa, and A. humilis. The presence of bacteria and timing of bacterial colonization was evaluated in gametes, swimming planulae, and newly settled polyps by fluorescence in situ hybridization (FISH) using general eubacterial probes and laser-scanning confocal microscopy. The coral species investigated in this study do not appear to transmit bacteria via their gametes, and bacteria are not detectable in or on the corals until after settlement and metamorphosis. This study suggests that mass-spawning corals do not acquire, or are not colonized by, detectable numbers of bacteria until after larval settlement and development of the juvenile polyp. This timing lays the groundwork for developing and testing new hypotheses regarding general regulatory mechanisms that control bacterial colonization and infection of corals, and how interactions among bacteria and juvenile polyps influence the structure of bacterial assemblages in corals.

Structure and function of the microbial community in a full-scale enhanced biological phosphorus removal plant

The structure and function of the microbial community in a full-scale enhanced biological phosphorus removal wastewater treatment plant (WWTP; Skagen) were investigated using the full-cycle rRNA approach, combined with ecophysiological studies. A total of 87 16S rRNA gene sequences were retrieved, and 78 operational taxonomic units were identified. Novel oligonucleotide probes were designed, and quantitative fluorescence in situ hybridization revealed that six hitherto undescribed probe-defined groups within the phylum Bacteroidetes (two groups), and classes Betaproteobacteria (two groups) and Gammaproteobacteria (two groups), were relatively abundant (>1% of total biovolume) in the Skagen WWTP and 10 other full-scale WWTPs with biological P removal. The most abundant was a group of rod-shaped Bacteroidetes attached to filamentous bacteria, which is distantly related to the genus Haliscomenobacter of the family Saprospiraceae, and comprised 9-19% of the bacterial biovolume in all the WWTPs investigated. The other five probe-defined groups were found in all WWTPs, but they were less abundant (1-6%). Two groups had a glycogen-accumulating phenotype and one Dechloromonas-related group had a polyphosphate-accumulating phenotype, and they were potentially all involved in denitrification. In total, about 81% of all bacteria hybridizing with the general eubacterial probe were detected in the Skagen WWTP by using clone- or group-specific probes, indicating that most members of the microbial community had been identified.

Host-Mediated Inflammation Disrupts the Intestinal Microbiota and Promotes the Overgrowth of Enterobacteriaceae

While the normal microbiota has been implicated as a critical defense against invading pathogens, the impact of enteropathogenic infection and host inflammation on intestinal microbial communities has not been elucidated. Using mouse models of Citrobacter rodentium, which closely mimics human diarrheal pathogens inducing host intestinal inflammation, and Campylobacter jejuni infection, as well as chemically and genetically induced models of intestinal inflammation, we demonstrate that host-mediated inflammation in response to an infecting agent, a chemical trigger, or genetic predisposition markedly alters the colonic microbial community. While eliminating a subset of indigenous microbiota, host-mediated inflammation supported the growth of either the resident or introduced aerobic bacteria, particularly of the Enterobacteriaceae family. Further, assault by an enteropathogen and host-mediated inflammation combined to significantly reduce the total numbers of resident colonic bacteria. These findings underscore the importance of intestinal microbial ecosystems in infectious colitis and noninfectious intestinal inflammatory conditions, such as inflammatory bowel disease.

Molecular Diversity, Cultivation, and Improved Detection by Fluorescent In Situ Hybridization of a Dominant Group of Human Gut Bacteria Related to Roseburia spp. or Eubacterium rectale

Phylogenetic analysis was used to compare 16S rRNA sequences from 19 cultured human gut strains of Roseburia and Eubacterium rectale with 356 related sequences derived from clone libraries. The cultured strains were found to represent five of the six phylotypes identified. A new oligonucleotide probe, Rrec584, and the previous group probe Rint623, when used in conjunction with a new helper oligonucleotide, each recognized an average of 7% of bacteria detected by the eubacterial probe Eub338 in feces from 10 healthy volunteers. Most of the diversity within this important group of butyrate-producing gut bacteria can apparently be retrieved through cultivation.

Design and Evaluation of 16S rRNA-Targeted Peptide Nucleic Acid Probes for Whole-Cell Detection of Members of the Genus Listeria

Six fluorescein-labeled peptide nucleic acid oligomers targeting Listeria-specific sequences on the 16S ribosomal subunit were evaluated for their abilities to hybridize to whole cells by fluorescence in situ hybridization (FISH). Four of these probes yielded weak or no fluorescent signals after hybridization and were not investigated further. The remaining two FISH-compatible probes, LisUn-3 and LisUn-11, were evaluated for their reactivities against 22 Listeria strains and 17 other bacterial strains belonging to 10 closely related genera. Hybridization with BacUni-1, a domain-specific eubacterial probe, was used as a positive control for target accessibility in both Listeria spp. and nontarget cells. RNase T1 treatment of select cell types was used to confirm that positive fluorescence responses were rRNA dependent and to examine the extent of nonspecific staining of nontarget cells. Both LisUn-3 and LisUn-11 yielded rapid, bright, and genus-specific hybridizations at probe concentrations of approximately 100 pmol ml(-1). LisUn-11 was the brightest probe and stained all six Listeria species. LisUn-3 hybridized with all Listeria spp. except for L. grayi, for which it had two mismatched bases. A simple ethanolic fixation yielded superior results with Listeria spp. compared to fixation in 10% buffered formalin and was applicable to all cell types studied. This study highlights the advantages of peptide nucleic acid probes for FISH-based detection of gram-positive bacteria and provides new tools for the rapid detection of Listeria spp. These probes may be useful for the routine monitoring of food production environments in support of efforts to control L. monocytogenes.

Rapid Diagnosis of Bacterial Meningitis by Real-Time PCR and Fluorescence In Situ Hybridization

Real-time PCR and fluorescence in situ hybridization (FISH) were evaluated as rapid methods for the diagnosis of bacterial meningitis and compared to standard diagnostic procedures. For PCR, a LightCycler approach was chosen, implementing eubacterial and specific PCR assays for the most relevant bacteria. For FISH, a similar probe set containing eubacterial and specific probes was composed of published and newly designed probes. Both methods were evaluated by use of cerebrospinal fluid (CSF) samples from patients with suspected bacterial meningitis. For all microscopy- and culture-positive samples (n = 28), the eubacterial PCR was positive. In addition, all identifiable pathogens were detected with specific PCR assays, according to an algorithm based on the Gram stain. The FISH method detected the pathogen in 13 of 18 positive samples. While the FISH method remained negative for all microscopy- and culture-negative samples (n = 113), the eubacterial PCR was positive for five of these samples. Sequencing of the amplicon revealed the presence of Neisseria meningitidis, Streptococcus agalactiae, and Haemophilus influenzae in three of these five samples. In addition, samples with discordant results by culture and microscopy were successfully investigated by PCR (10 samples) and FISH (5 samples). In conclusion, PCR is a highly sensitive tool for rapid diagnosis of bacterial meningitis. FISH is less sensitive but is useful for the identification of CSF samples showing bacteria in the Gram stain. Based on our results, an approach for laboratory diagnosis of meningitis including PCR and FISH is discussed.

Development and field application of a molecular probe for the primary pathogen of the coral disease white plague type II.

One of the current problems in the field of coral disease research is that of tracking coral pathogens in the natural environment. A promising method to do this is by use of pathogen-specific molecular probes. However, this approach has been little used to date. We constructed, and validated in the laboratory, a fluorochrome-labeled molecular probe specific to Aurantimonas coralicida, the bacterial pathogen of the Caribbean coral disease white plague type II (WPIl). We then used the probe to test field samples of diseased coral tissue for the presence of this pathogen. Probe design was based on a unique subset (25 nucleotides) of the complete l6S rRNA gene sequence derived from a pure culture of the pathogen. The pathogen-specific probe was labeled with the fluorochrome GreenStar* FITC (fluorescein isothiocyanate, GeneDetect Ltd, New Zealand). As a control, we used the universal eubacterial probe EUB 338, labeled with a different fluorochrome (TRITC, tetra-methylrhodamine isothiocyanate). Both probes were applied to laboratory samples of pure cultures of bacteria, and field samples collected from the surface of the disease line of corals exhibiting signs of white plague (types I and II), healthy controls, and corals with an uncharacterized disease ("patchy necrosis"). All samples were analyzed using fluorescence in situ hybridization (FISH). We have determined that the probe is specific to our laboratory culture of the coral pathogen, and does not react with other bacterial species (the eubacterial probe does). The WPII pathogen was detected in association with diseased coral samples collected from coral colonies on reefs of the Bahamas (n= 9 samples) exhibiting signs of both WPI and WPII. Diseased (and healthy) tissue samples (n- 4) from corals exhibiting signs of "patchy necrosis" were also assayed. In this case the results were negative, indicating that the same pathogen is not involved in the two diseases. Incorporation and use of pathogen-specific probes can significantly expand our knowledge of the etiology of coral diseases.

Hybridisation of picoeukaryotes by eubacterial probes is widespread in the marine environment

Most general and group-specific eubacterial probes hybridised picoeukaryotes in coastal waters (Brittany, France) and cultures of the dominant picoeukaryotes from this environment (Micromonas pusilla and Pelagomonas calceolata). This is either because they matched the 16S rRNA from organelles or because of the presence of symbiotic or antagonist intracellular bacteria. The general eubacterial probe (EUB338) hybridised 84% of the picoeukaryotes, while the group-specific probes hybridised 3, 16, 10 and 34% of the picoeukaryotes for cyanobacteria (CYA664), alphaproteobacteria (ALF968), gamma-proteobacteria (GAM42a) and Cytophaga-Flavo-Bacteria (CF319), respectively. The results show that the hybridisation of eukaryote 16S rRNA by prokaryote probes can lead to significant errors in prokaryote counts, in particular for less well-represented groups such as cyanobacteria, with errors of 17% in the studied sample. In addition, we revealed for the first time at this scale that up to 44% of the picoeukaryotes contained intracellular prokaryotes. This finding might have serious implications for understanding the functioning of the microbial loop. Finally, because SSU rRNA databases have significantly been extended in recent years, we showed that the probe PLA886, which targets Planctomycete 16S rRNA, labelled 87% of the picoeukaryotes by hybridising their 18S rRNA. Consequently, the design of this probe should be refined for future studies, and the presence of similar changes in probe specificity should be checked regularly when using hybridisation-based techniques.

Detection and quantitative estimation of Dehalococcoides spp. in a dechlorinating bioreactor by a combination of fluorescent in situ hybridisation (FISH) and kinetic analysis.

The unique capacity of Dehalococcoides ethenogenes of completely dechlorinating the common groundwater pollutant tetrachloroethene (PCE) to the harmless ethene makes this microorganism very attractive for application in natural or engineered bioremediation systems. In this study, the qualitative and quantitative determination of Dehalococcoides spp. in a lab-scale bioreactor was performed based on the combination of fluorescent in situ hybridisation (FISH) for specific detection, and kinetic batch tests at non-limiting hydrogen and PCE concentration for quantitative determination. The dechlorinating bioreactor was operated at a high and constant PCE loading rate of 255 micromol PCE [g volatile suspended solids (VSS)](-1) day(-1). Pale coccoid cells resembling the distinctive morphotype of D. ethenogenes were present in the microbial culture. These cocci hybridised with both eubacterial probes and the Dhe1259t probe recently designed for detecting Dehalococcoides spp. Positive hybridisation was also observed when the DHC1377 reverse primer was used as a specific probe and applied to the dechlorinating microbial consortium. The maximum dechlorination rate obtained under non-limiting hydrogen and PCE concentrations was 3.22 +/- 0.08 mmol Cl(-) l(-1 )day(-1). From the specific activity of D. ethenogenes [i.e. 0.055 +/- 0.008 mmol Cl(-) (mg VSS)(-1) day(-1)], as reported from pure culture study, this observed maximum rate corresponded to a concentration of this bacterium in the mixed liquor of the bioreactor of 59.0+/-10.4 mg VSS.l(-1) (41.5+/-11.2% of overall VSS). This calculated relative abundance of D. ethenogenes was in agreement with the percentage of methanol (in terms of reducing equivalents) channeled to reductive dechlorination (approximately 30%) supporting the assumption that most reductive dechlorination was actually due to this microorganism.

Development of alternate ssu-rRNA probing strategies for characterizing aquatic microbial communities

Plastids in phytoplankton retain prokaryote-like DNA sequences that may generate false-positive signals from eubacterial small subunit (ssu) rRNA oligonucleotide probes, resulting in the overestimation of bacterial activity in aquatic microbial communities. To assess the extent of possible plastid-associated binding to eubacterial signals, we performed an extensive database search, flask experiments using algal and cyanobacterial pure cultures, and field trials on five common eubacterial probes: S-D-Bact-008-a-A-19, S-D-Bact-338-a-A-18, S-D-Bact-785-a-A-19, S-D-Bact-927-a-A-17, and S-D-Bact-1088-a-A-20. The database search and laboratory tests showed significant potential for binding among most bacterial probes and organelle ssu-rRNA. However, we propose two probing strategies to overcome this problem. First, one could use Bact-785 and Bact-338 in tandem, with the plastid component being estimated as the difference between the two signals (Bact-338 has ~70% overlap with known plastid sequences). Alternately, one might use Bact-338 as the primary eubacterial probe, but then use Cyan-785-a-A-19 (a probe that binds significantly to plastid rRNA) to correct for the plastid-associated false-positive signal. Both strategies would use a eukaryotic probe (S-D-Euca-1379-a-A-16) and Cyan-785-b-A-19 (a probe for most cyanobacteria) to further segregate rRNA signals. Trials were successfully performed using the strategies on samples from a recent field study.

Leech mycetome endosymbionts are a new lineage of alphaproteobacteria related to the Rhizobiaceae

Mycetomal organs attached to the esophagus of hematophagous leeches which are known to harbor endosymbiotic bacteria were removed from three species in the leech family Glossiphoniidae. Anatomical observations indicated that placobdellid mycetomes are paired and caecate, inserting into the esophagus posterior to the proboscis. Light and electron microscopy demonstrated that there is a single layer of mycetome epithelial cells harboring Gram-negative rods and that these epithelial cells are ultrastructurally distinct from neighboring esophageal epithelial cells. Fluorescent in situ hybridization with eubacterial and alphaproteobacterial probes localized the bacteria solely to the mycetomes both in adult and in unfed juvenile leeches whereas a gammaproteobacterial probe did not yield a bound fluorescencent signal. DNA was isolated from these tissues and subjected to PCR amplification using bacteria-specific primers for 16S and 23S rDNA. Results from sequencing the amplification products and phylogenetic analysis with other Alphaproteobacteria revealed that the bacteria resident in these organs comprise a new genus of Alphaproteobacteria, Reichenowia n. gen., closely related to the nitrogen-fixing, nodule-forming Rhizobiaceae. The three bacterial strains, though different from each other were each other’s closest relatives, suggesting a history of close coevolution with their leech hosts.

Intracellular bacteria associated with the ascidian Ecteinascidia turbinata : phylogenetic and in situ hybridisation analysis

The ascidian Ecteinascidia turbinata (Herdman) is a colonial sea squirt found in the Caribbean and Mediterranean Seas. In the present study, the bacterial complement of E. turbinata has been assessed by 16S rRNA gene analysis and the most commonly occurring strains identified by restriction fragment length polymorphism. Three strains were found to predominate using this approach, with one representing >50% of clones from both larval and adult material. The 16S rRNA gene sequence of the most commonly occurring strain did not match with any known bacterial sequences and could only be assigned to the γ-proteobacteria subdivision. The two other frequently occurring strains were assigned to the Mollicutes. In situ hybridisation analysis with eubacterial probes to 16S rRNA revealed the presence of apparently endosymbiotic bacteria in adult and larval tissue, and electron microscopy confirmed the presence of putative bacteriocytes in the larval tissue. The presence of the same bacteria in the brooded larvae suggested that they were vertically transmitted from parent to offspring. Further hybridisation using a novel probe designed to be specific to the 16S rRNA sequence of the dominant strain, highlighted the same cell types as that revealed by the eubacterial probe. The results suggest that the bacteria represent a novel strain, denoted "Candidatus Endoecteinascidia frumentensis", and that they may have an important role in the biology of E. turbinata.